ALK4.ActRIIB heteromultimers

ABSTRACT

In certain aspects, the disclosure provides soluble heteromeric polypeptide complexes comprising an extracellular domain of an ALK4 receptor and an extracellular domain of ActRIIB. In certain aspects, such soluble ALK4:ActRIIB complexes may be used to regulate (promote or inhibit) growth of tissues or cells including, for example, muscle, bone, cartilage, fat, neural tissue, tumors, and/or cancerous cells. In certain aspects, such ALK4:ActRIIB complexes are can be used to improve muscle formation, bone formation, metabolic parameters, and disorders associated with these tissues, cellular networks, kidney, and endocrine systems.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority from U.S. ProvisionalApplication No. 62/404,727, filed Oct. 5, 2016, and U.S. ProvisionalApplication No. 62/510,417, filed May 24, 2017. The specifications ofthe foregoing applications are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 16, 2017, isnamed 1848179-0002-117-101_SL.txt and is 150,252 bytes in size.

BACKGROUND OF THE INVENTION

The transforming growth factor-beta (TGF-beta) superfamily contains avariety of growth factors that share common sequence elements andstructural motifs. These proteins are known to exert biological effectson a large variety of cell types in both vertebrates and invertebrates.Members of the superfamily perform important functions during embryonicdevelopment in pattern formation and tissue specification and caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis,neurogenesis, and epithelial cell differentiation. The family is dividedinto two general phylogenetic clades: the more recently evolved membersof the superfamily, which includes TGF-betas, activins, and nodal andthe clade of more distantly related proteins of the superfamily, whichincludes a number of BMPs and GDFs [Hinck (2012) FEBS Letters586:1860-1870]. TGF-beta family members have diverse, oftencomplementary biological effects. By manipulating the activity of amember of the TGF-beta family, it is often possible to cause significantphysiological changes in an organism. For example, the Piedmontese andBelgian Blue cattle breeds carry a loss-of-function mutation in the GDF8(also called myostatin) gene that causes a marked increase in musclemass [Grobet et al. (1997) Nat Genet 17(1):71-4]. Furthermore, inhumans, inactive alleles of GDF8 are associated with increased musclemass and, reportedly, exceptional strength [Schuelke et al. (2004) NEngl J Med 350:2682-8].

Changes in fibrosis, muscle, bone, fat, and other tissues may beachieved by enhancing or inhibiting intracellular signaling (e.g., SMAD1, 2, 3, 5, and/or 8) that is mediated by ligands of the TGF-betafamily. Thus, there is a need for agents that regulate the activity ofvarious ligands of the TGF-beta superfamily.

SUMMARY OF THE INVENTION

As described herein, it has been discovered that an ALK4:ActRIIBheterodimer protein is a unique antagonist of ligands of the TGF-betasuperfamily, exhibiting a different ligand-binding profile/selectivitycompared to corresponding ActRIIB and ALK4 homodimers. In particular, anexemplary ALK4:ActRIIB heterodimer displays enhanced binding to activinB compared to either homodimer, retains strong binding to activin A,GDF8, and GDF11 as observed with ActRIIB homodimer, and exhibitssubstantially reduced binding to BMP9, BMP10, and GDF3. In fact, theALK4:ActRIIB heterodimer displays low to no observable affinity forBMP9, whereas this ligand binds strongly to ActRIIB homodimer. See FIG.4. These results therefore demonstrate that ALK4:ActRIIB heterodimersare a more selective antagonists (inhibitors) of certain ligands of theTGF-beta superfamily compared to ActRIIB homodimers. Accordingly, anALK4:ActRIIB heterodimer will be more useful than an ActRIIB homodimerin certain applications where such selective antagonism is advantageous.Examples include therapeutic applications where it is desirable toantagonize one or more of activin (e.g., activin A, activin B, activinAB, activin AC), GDF8, and GDF11 with decreased antagonism of one ormore of BMP9, BMP10, and GDF3.

Moreover, ALK4:ActRIIB heterodimer produced certain biological effectsstrikingly similar to those of an ActRIIB homodimer despite differentialligand selectivity of the two complexes. For example, ALK4:ActRIIBheterodimer exerts beneficial anabolic effects on skeletal muscle andbone as well as catabolic effects on adipose tissue, very similar tothose of an ActRIIB-Fc homodimer. However, unlike ActRIIB homodimer,ActRIIB:ALK4 heterodimer exhibits only low-affinity or transient bindingto BMP9 and BMP10 and so should have little to no concurrent inhibitionon processes mediated by those ligands, such as angiogenesis. This novelselectivity may be useful, for example, in treating patients in need ofstimulatory effects on muscle and bone, and/or inhibitory effects onfat, but not in need of altered angiogenesis. In addition, ALK4:ActRIIBheterodimer had various beneficial effects in a mouse model of kidneydisease, particularly on treating or preventing kidney damage,inflammation, and fibrosis. Therefore, while not wishing to be bound toa particular mechanisms of action, it is expected that ALK4:ActRIIBheteromultimers, as well as variants thereof, that bind to/inhibit atleast one or more of activin (e.g., activin A, activin B, activin AB,and activin AC), GDF8, and/or GDF11 will be useful agents for promotingbeneficial anabolic effects on skeletal muscle and bone, cataboliceffects on adipose tissue, and beneficial effects on kidney disease.

Therefore, the present disclosure relates, in part, to heteromultimercomplexes (heteromultimers) comprising at least one ALK4 polypeptide andat least one ActRIIB polypeptide (ALK4:ActRIIB heteromultimers).Preferably, ALK4 polypeptides comprise a ligand-binding domain of anALK4 receptor, for example, a portion of the ALK4 extracellular domain.Similarly, ActRIIB polypeptides generally comprise a ligand-bindingdomain of an ActRIIB receptor, for example, a portion of the ActRIIBextracellular domain. Preferably, such ALK4 and ActRIIB polypeptides, aswell as resultant heteromultimers thereof, are soluble. Preferably, suchALK4 and ActRIIB polypeptides, as well as resultant heteromultimersthereof, are recombinant proteins. Preferably, such ALK4 and ActRIIBpolypeptides, as well as resultant heteromultimers thereof, are isolatedproteins.

In certain aspects, an ALK4:ActRIIB heteromultimer comprises an ALK4domain comprising an amino acid sequence that is at least 70% identicalto a polypeptide that begins at any one of amino acids 24-34 of SEQ IDNO: 9 (e.g., amino acids 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34)and ends at any one of amino acids 101-126 of SEQ ID NO: 9 (e.g., aminoacids 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, and 126).For example, ALK4:ActRIIB heteromultimers may comprise an amino acidsequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to aminoacids 34-101 of SEQ ID NO: 9. In some embodiments, ALK4:ActRIIBheteromultimers may comprise an amino acid sequence that is at least70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to amino acids 24-126 of SEQ IDNO: 9. In some embodiments, ALK4:ActRIIB heteromultimers may comprise anALK4 amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 10.

In other aspects, an ALK4:ActRIIB heteromultimer comprises an ALK4domain comprising an amino acid sequence that is at least 70% identicalto a polypeptide that begins at any one of amino acids 24-34 of SEQ IDNO: 19 (e.g., amino acids 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, and34) and ends at any one of amino acids 101-126 of SEQ ID NO: 19 (e.g.,amino acids 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, and126). For example, ALK4:ActRIIB heteromultimers may comprise an aminoacid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical toamino acids 34-101 of SEQ ID NO: 19. In some embodiments, ALK4:ActRIIBheteromultimers may comprise an amino acid sequence that is at least70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to amino acids 24-126 of SEQ IDNO: 19. In some embodiments, ALK4:ActRIIB heteromultimers may comprisean ALK4 amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 20.

In certain aspects, an ALK4:ActRIIB heteromultimer comprises an ActRIIBdomain comprising an amino acid sequence that is at least 70% identicalto a polypeptide that begins at any one of amino acids 20-29 of SEQ IDNO: 1 (e.g., amino acids 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29) andends at any one of amino acids 109-134 (109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, and 134) of SEQ ID NO: 1. For example,ALK4:ActRIIB heteromultimers may comprise an amino acid sequence that isat least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 29-109 ofSEQ ID NO: 1. In some embodiments, ALK4:ActRIIB heteromultimers maycomprise an amino acid sequence that is at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to amino acids 25-131 of SEQ ID NO: 1. In someembodiments, ALK4:ActRIIB heteromultimers may comprise an ActRIIB aminoacid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical toSEQ ID NO: 2. In some embodiments, ALK4:ActRIIB heteromultimers maycomprise an ActRIIB amino acid sequence that is at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 3. In some embodiments,ALK4:ActRIIB heteromultimers do not comprise an ActRIIB polypeptidecomprising an acidic amino acid (e.g., the naturally occurring aminoacids E or D or an artificial acidic amino acid) at the positioncorresponding to L79 of SEQ ID NO: 1.

In other aspects, an ALK4:ActRIIB heteromultimer comprises an ActRIIBdomain comprising an amino acid sequence that is at least 70% identicalto a polypeptide that begins at any one of amino acids 20-29 of SEQ IDNO: 4 (e.g., amino acids 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29) andends at any one of amino acids 109-134 (109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, and 134) of SEQ ID NO: 4. For example,ALK4:ActRIIB heteromultimers may comprise an amino acid sequence that isat least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 29-109 ofSEQ ID NO: 4. In some embodiments, ALK4:ActRIIB heteromultimers maycomprise an amino acid sequence that is at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to amino acids 25-131 of SEQ ID NO: 4. In someembodiments, ALK4:ActRIIB heteromultimers may comprise an ActRIIB aminoacid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical toSEQ ID NO: 5. In some embodiments, ALK4:ActRIIB heteromultimers maycomprise an ActRIIB amino acid sequence that is at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 6. In some embodiments,ALK4:ActRIIB heteromultimers do not comprise an ActRIIB polypeptidecomprising an acidic amino acid (e.g., the naturally occurring aminoacids E or D or an artificial acidic amino acid) at the positioncorresponding to L79 of SEQ ID NO: 1.

As described herein, ALK4:ActRIIB heteromultimer structures include, forexample, heterodimers, heterotrimers, heterotetramers, heteropentamers,and higher order heteromultimer complexes. See, e.g., FIG. 6. In certainpreferred embodiments, ALK4:ActRIIB heteromultimers are heterodimers.

In certain aspects, ALK4 and/or ActRIIB polypeptides may be fusionproteins. For example, in some embodiments, an ALK4 polypeptide may be afusion protein comprising an ALK4 polypeptide domain and one or moreheterologous (non-ALK4) polypeptide domains (e.g., ALK4-Fc fusionproteins). Similarly, in some embodiments, an ActRIIB polypeptide may bea fusion protein comprising an ActRIIB polypeptide domain and one ormore heterologous (non-ActRIIB) polypeptide domains (ActRIIB-Fc fusionproteins).

In some embodiments, ALK4 polypeptides are fusion proteins that comprisean Fc domain of an immunoglobulin. Similarly, in some embodiments,ActRIIB polypeptides are fusion proteins that comprise an Fc domain ofan immunoglobulin. Traditional Fc fusion proteins and antibodies areexamples of unguided interaction pairs, whereas a variety of engineeredFc domains have been designed as asymmetric interaction pairs [Spiess etal (2015) Molecular Immunology 67(2A): 95-106]. Therefore, a firstmember and/or a second member of an interaction pair described hereinmay comprise a constant domain of an immunoglobulin, including, forexample, the Fc portion of an immunoglobulin. For example, a firstmember of an interaction pair may comprise an amino acid sequence thatis derived from an Fc domain of an IgG (IgG1, IgG2, IgG3, or IgG4), IgA(IgA1 or IgA2), IgE, or IgM immunoglobulin. Such immunoglobulin domainsmay comprise one or more amino acid modifications (e.g., deletions,additions, and/or substitutions) that promote ALK4:ActRIIBheteromultimer formation. Similarly, a second member of an interactionpair may comprise an amino acid sequence that is derived from an Fcdomain of an IgG (IgG1, IgG2, IgG3, or IgG4), IgA (IgA1 or IgA2), IgE,or IgM. Such immunoglobulin domains may comprise one or more amino acidmodifications (e.g., deletions, additions, and/or substitutions) thatpromote ALK4:ActRIIB heteromultimer formation. For example, the secondmember of an interaction pair may comprise, consist essentially of, orconsist of an amino acid sequence that is at least 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical toany one of SEQ ID NOs: 23-37. In some embodiments, a first member and asecond member of an interaction pair comprise Fc domains derived fromthe same immunoglobulin class and subtype. In other embodiments, a firstmember and a second member of an interaction pair comprise Fc domainsderived from different immunoglobulin classes or subtypes.

In certain aspects, the disclosure relates to ALK4:ActRIIBheteromultimers comprising at least one ALK4-Fc fusion protein and atleast one ActRIIB-Fc fusion protein wherein the ALK4-Fc fusion proteincomprises one or more amino acid modifications (e.g., amino acidsubstitution, cationization, deamination, carboxyl-terminal amino acidheterogeneity, phosphorylation, and glycosylation) that alter theisoelectric point (pI) of the ALK4-Fc fusion protein and/or theActRIIB-Fc fusion protein comprises one or more amino acid modificationsthat alter the pI of the ActRIIB-Fc fusion protein. In some embodiments,the one or more amino acid modifications in the ALK4-Fc fusion proteinconfers increased difference in pIs between the ALK4-Fc fusion proteinand the ActRIIB-Fc fusion protein. In other embodiments, the one or moreamino acid modifications in the ActRIIB-Fc fusion protein confersincreased difference in pIs between the ActRIIB-Fc fusion protein andthe ALK4-Fc fusion protein. In still other embodiments the one or moreamino acid modifications in the ALK4-Fc fusion protein confers increaseddifference in pIs between the ALK4-Fc fusion protein and the ActRIIB-Fcfusion protein, and the one or more amino acid modifications in theActRIIB-Fc fusion protein confers increased difference in pIs betweenthe ActRIIB-Fc fusion protein and the ALK4-Fc fusion protein. In someembodiments, the ALK4-Fc fusion protein comprises one or more amino acidmodifications that alter pI by at least 0.1 (e.g., by at least 0.1, 0.2,0.3, 0.4, 0.5, 0.7, 0.8. 0.9, 1.0, 1.3, 1.5, 1.7, 2.0, 2.3, 2.5, 2.7,3.0, 3.3, 3.5, 3.7, or at least by 4.0). In some embodiments, theActRIIB-Fc fusion protein comprises one or more amino acid modificationsthat alter pI by at least 0.1 (e.g., by at least 0.1, 0.2, 0.3, 0.4,0.5, 0.7, 0.8. 0.9, 1.0, 1.3, 1.5, 1.7, 2.0, 2.3, 2.5, 2.7, 3.0, 3.3,3.5, 3.7, or at least by 4.0). In some embodiments, the ALK4-Fc fusionprotein comprises one or more amino acid modifications that alter pI byat least 0.1 (e.g., by at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 0.8. 0.9,1.0, 1.3, 1.5, 1.7, 2.0, 2.3, 2.5, 2.7, 3.0, 3.3, 3.5, 3.7, or at leastby 4.0) and the ActRIIB-Fc fusion protein comprises one or more aminoacid modifications that alter pI by at least 0.1 (e.g., by at least 0.1,0.2, 0.3, 0.4, 0.5, 0.7, 0.8. 0.9, 1.0, 1.3, 1.5, 1.7, 2.0, 2.3, 2.5,2.7, 3.0, 3.3, 3.5, 3.7, or at least by 4.0). In some embodiments, theALK4-Fc fusion protein and the ActRIIB-Fc fusion protein have at least a0.7 difference in pI (e.g., at least 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3.1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or at least4.0 or more difference in pI).

In certain aspects, an ALK4:ActRIIB heteromultimer of the disclosurecomprises an ALK4-Fc fusion protein comprising one or more amino acidmodifications that increase the pI of the ALK4-Fc fusion protein; and anActRIIB-Fc fusion protein comprising one or more amino acidmodifications that decrease the pI of the ActRIIB-Fc fusion protein. Forexample, an ALK4-Fc fusion protein may be modified by substituting oneor more neutral or negatively charged amino acids with one or morepositively charged amino acids [e.g., an arginine (R), lysine (K), orhistidine (H)]. Similarly, an ActRIIB-Fc fusion protein may be modifiedby substituting one or more neutral or positively charged amino acidswith one or more negatively charged amino acids [e.g., aspartic acid (E)or glutamic acid (D)]. In some embodiments, the ALK4-Fc fusion proteinFc domain is an IgG1 Fc domain that comprises one or more amino acidmodifications that alter the pI of the ALK4-Fc fusion protein. In someembodiments, the ALK4-Fc fusion protein IgG1 Fc domain comprises anamino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acidsequence of SEQ ID NO: 31. In some embodiments, the ALK4-Fc fusionprotein IgG1 Fc domain comprises one or more amino acid substitutionsselected from: a) an amino acid substitution at the positioncorresponding to N162 of SEQ ID NO: 31; b) an amino acid substitution atthe position corresponding to D179 of SEQ ID NO: 31; and c) an aminoacid substitution at the position corresponding to N162 of SEQ ID NO: 31and an amino acid substitution at the position corresponding to D179 ofSEQ ID NO: 31. In some embodiments, the ALK4-Fc fusion protein IgG1 Fcdomain comprises one or more amino acid substitutions selected from: a)an arginine, lysine, or histidine substitution at the positioncorresponding to N162 of SEQ ID NO: 31 (N162R, N162K, or N162H); b) anarginine, lysine, or histidine substitution at the positioncorresponding to D179 of SEQ ID NO: 31 (D179R, D179K, or D179H); and c)an arginine, lysine, or histidine substitution at the positioncorresponding to N162 of SEQ ID NO: 31 (N162R, N162K. or N162H) and anarginine, lysine, or histidine substitution at the positioncorresponding to D179 of SEQ ID NO: 31 (D179R, D179K. or D179H). In someembodiments, the ALK4-Fc fusion protein IgG1 Fc domain comprises one ormore amino acid substitutions selected from: a) an arginine substitutionat the position corresponding to N162 of SEQ ID NO: 31 (N162R); b) anarginine substitution at the position corresponding to D179 of SEQ IDNO: 31 (D179R); and c) an arginine substitution at the positioncorresponding to N162 of SEQ ID NO: 31 (N162R) and an argininesubstitution at the position corresponding to D179 of SEQ ID NO: 31(D179R). In some embodiments, the ALK4-Fc fusion protein Fc domain is anIgG2 Fc domain that comprises one or more amino acid modifications thatalter the pI of the ALK4-Fc fusion protein. In some embodiments, theALK4-Fc fusion protein IgG2 Fc domain comprises an amino acid sequencethat is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more identical to the amino acid sequence of SEQ ID NO: 32.In some embodiments, the ALK4-Fc fusion protein IgG2 Fc domain comprisesone or more amino acid substitutions selected from: a) an amino acidsubstitution at the position corresponding to N160 of SEQ ID NO: 32; b)an amino acid substitution at the position corresponding to D177 of SEQID NO: 32; and c) an amino acid substitution at the positioncorresponding to N160 of SEQ ID NO: 32 and an amino acid substitution atthe position corresponding to D177 of SEQ ID NO: 32. In someembodiments, the ALK4-Fc fusion protein IgG2 Fc domain comprises one ormore amino acid substitutions selected from: a) an arginine, lysine, orhistidine substitution at the position corresponding to N160 of SEQ IDNO: 32 (N160R, N160K, or N160H); b) an arginine, lysine, or histidinesubstitution at the position corresponding to D177 of SEQ ID NO: 32(D177R, D177K, or D177H); and c) an arginine, lysine, or histidinesubstitution at the position corresponding to N160 of SEQ ID NO: 32(N160R, N160K, or N160H) and an arginine, lysine, or histidinesubstitution at the position corresponding to D177 of SEQ ID NO: 32(D177R, D177K. or D177H). In some embodiments, the ALK4-Fc fusionprotein Fc domain is an IgG3 Fc domain that comprises one or more aminoacid modifications that alter the pI of the ALK4-Fc fusion protein. Insome embodiments, the ALK4-Fc fusion protein IgG3 Fc domain comprises anamino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acidsequence of SEQ ID NO: 33. In some embodiments, the ALK4-Fc fusionprotein IgG3 Fc domain comprises one or more amino acid substitutionsselected from: a) an amino acid substitution at the positioncorresponding to S169 of SEQ ID NO: 33; b) an amino acid substitution atthe position corresponding to D186 of SEQ ID NO: 33; and c) an aminoacid substitution at the position corresponding to S169 of SEQ ID NO: 33and an amino acid substitution at the position corresponding to D186 ofSEQ ID NO: 33. In some embodiments, the ALK4-Fc fusion protein IgG3 Fcdomain comprises one or more amino acid substitutions selected from: a)an arginine, lysine, or histidine substitution at the positioncorresponding to S169 of SEQ ID NO: 33 (S169R, S169K, or S169H); b) anarginine, lysine, or histidine substitution at the positioncorresponding to D186 of SEQ ID NO: 33 (D186R, D186K, or D186H); and c)an arginine, lysine, or histidine substitution at the positioncorresponding to S169 of SEQ ID NO: 33 (S169R, S169K, or S169H) and anarginine, lysine, or histidine substitution at the positioncorresponding to D186 of SEQ ID NO: 33 (D186R, D186K, or D186H). In someembodiments, the ALK4-Fc fusion protein Fc domain is an IgG4 Fc domainthat comprises one or more amino acid modifications that alter the pI ofthe ALK4-Fc fusion protein. In some embodiments, the ALK4-Fc fusionprotein IgG4 Fc domain comprises an amino acid sequence that is at least75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 35. In someembodiments, the ALK4-Fc fusion protein IgG4 Fc domain comprises one ormore amino acid substitutions selected from: a) an amino acidsubstitution at the position corresponding to N166 of SEQ ID NO: 35; b)an amino acid substitution at the position corresponding to D183 of SEQID NO: 35; and c) an amino acid substitution at the positioncorresponding to N166 of SEQ ID NO: 35 and an amino acid substitution atthe position corresponding to D183 of SEQ ID NO: 35. In someembodiments, the ALK4-Fc fusion protein IgG4 Fc domain comprises one ormore amino acid substitutions selected from: a) an arginine, lysine, orhistidine substitution at the position corresponding to N166 of SEQ IDNO: 35 (N166R, N166K, or N166H); b) an arginine, lysine, or histidinesubstitution at the position corresponding to D183 of SEQ ID NO: 35(D183R, D183K, or D183H); and c) an arginine, lysine, or histidinesubstitution at the position corresponding to N166 of SEQ ID NO: 35(N166R, N166K, or N166H) and an arginine, lysine, or histidinesubstitution at the position corresponding to D183 of SEQ ID NO: 32(D183R, D183K. or D183H). In some embodiments, the ActRIIB-Fc fusionprotein Fc domain is an IgG1 Fc domain that comprises one or more aminoacid modifications that alter the pI of the ActRIIB-Fc fusion protein.In some embodiments, the ActRIIB-Fc fusion protein IgG1 Fc domaincomprises an amino acid sequence that is at least 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to theamino acid sequence of SEQ ID NO: 31. In some embodiments, theActRIIB-Fc fusion protein IgG1 Fc domain comprises one or more aminoacid substitutions selected from: a) an amino acid substitution at theposition corresponding to K138 of SEQ ID NO: 31; b) an amino acidsubstitution at the position corresponding to K217 of SEQ ID NO: 31; andc) an amino acid substitution at the position corresponding to K138 ofSEQ ID NO: 31 and an amino acid substitution at the positioncorresponding to K217 of SEQ ID NO: 31. In some embodiments, theActRIIB-Fc fusion protein IgG1 Fc domain comprises one or more aminoacid substitutions selected from: a) an aspartic acid or glutamic acidsubstitution at the position corresponding to K138 of SEQ ID NO: 31(K138E or K138D); b) an aspartic acid or glutamic acid substitution atthe position corresponding to K217 of SEQ ID NO: 31 (K217E or K217D);and c) an aspartic acid or glutamic acid substitution at the positioncorresponding to K138 of SEQ ID NO: 31 (K138E or K138D) and an asparticacid or glutamic acid substitution at the position corresponding to K217of SEQ ID NO: 31 (K217E or K217D). In some embodiments, the ALK4-Fcfusion protein IgG1 Fc domain comprises one or more amino acidsubstitutions selected from: a) a glutamic acid substitution at theposition corresponding to K138 of SEQ ID NO: 31 (K138E); b) an asparticacid substitution at the position corresponding to K217 of SEQ ID NO: 31(K217D); and c) a glutamic acid substitution at the positioncorresponding to K138 of SEQ ID NO: 31 (K138E) and an aspartic acidsubstitution at the position corresponding to K217 of SEQ ID NO: 31(K217D). In some embodiments, the ActRIIB-Fc fusion protein Fc domain isan IgG2 Fc domain that comprises one or more amino acid modificationsthat alter the pI of the ActRIIB-Fc fusion protein. In some embodiments,the ActRIIB-Fc fusion protein IgG2 Fc domain comprises an amino acidsequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQID NO: 32. In some embodiments, the ActRIIB-Fc fusion protein IgG2fusion Fc domain comprises one or more amino acid substitutions selectedfrom: a) an amino acid substitution at the position corresponding toK136 of SEQ ID NO: 32; b) an amino acid substitution at the positioncorresponding to K215 of SEQ ID NO: 32; and c) an amino acidsubstitution at the position corresponding to K136 of SEQ ID NO: 32 andan amino acid substitution at the position corresponding to K215 of SEQID NO: 32. In some embodiments, the ActRIIB-Fc fusion protein IgG2 Fcdomain comprises one or more amino acid substitutions selected from: a)an aspartic acid or glutamic acid substitution at the positioncorresponding to K136 of SEQ ID NO: 32 (K136E or K136D); b) an asparticacid or glutamic acid substitution at the position corresponding to K215of SEQ ID NO: 32 (K215E or K215D); and c) an aspartic acid or glutamicacid substitution at the position corresponding to K136 of SEQ ID NO: 32(K136E or K136D) and an aspartic acid or glutamic acid substitution atthe position corresponding to K215 of SEQ ID NO: 32 (K215E or K215D). Insome embodiments, the ActRIIB-Fc fusion protein Fc domain is an IgG3 Fcdomain that comprises one or more amino acid modifications that alterthe pI of the ActRIIB-Fc fusion protein. In some embodiments, theActRIIB-Fc fusion protein IgG3 Fc domain comprises an amino acidsequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQID NO: 33. In some embodiments, the ActRIIB-Fc fusion protein IgG3fusion Fc domain comprises one or more amino acid substitutions selectedfrom: a) an amino acid substitution at the position corresponding toK145 of SEQ ID NO: 33; b) an amino acid substitution at the positioncorresponding to K224 of SEQ ID NO: 33; and c) an amino acidsubstitution at the position corresponding to K145 of SEQ ID NO: 33 andan amino acid substitution at the position corresponding to K224 of SEQID NO: 33. In some embodiments, the modified ActRIIB-Fc fusion proteinIgG3 Fc domain comprises one or more amino acid substitutions selectedfrom: a) an aspartic acid or glutamic acid substitution at the positioncorresponding to K145 of SEQ ID NO: 33 (K145E or K145D); b) an asparticacid or glutamic acid substitution at the position corresponding to K224of SEQ ID NO: 33 (K224E or K224D); and c) an aspartic acid or glutamicacid substitution at the position corresponding to K145 of SEQ ID NO: 33(K145E or K145D) and an aspartic acid or glutamic acid substitution atthe position corresponding to K224 of SEQ ID NO: 33 (K224E or K224D). Insome embodiments, the ActRIIB-Fc fusion protein Fc domain is an IgG4 Fcdomain that comprises one or more amino acid modifications that alterthe pI of the ActRIIB-Fc fusion protein. In some embodiments, theActRIIB-Fc fusion protein IgG4 Fc domain comprises an amino acidsequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQID NO: 35. In some embodiments, the ActRIIB-Fc fusion protein IgG4fusion Fc domain comprises one or more amino acid substitutions selectedfrom: a) an amino acid substitution at the position corresponding toK142 of SEQ ID NO: 35; b) an amino acid substitution at the positioncorresponding to K221 of SEQ ID NO: 35; and c) an amino acidsubstitution at the position corresponding to K142 of SEQ ID NO: 35 andan amino acid substitution at the position corresponding to K221 of SEQID NO: 35. In some embodiments, the ActRIIB-Fc fusion protein IgG4 Fcdomain comprises one or more amino acid substitutions selected from: a)an aspartic acid or glutamic acid substitution at the positioncorresponding to K142 of SEQ ID NO: 35 (K142E or K142D); b) an asparticacid or glutamic acid substitution at the position corresponding to K221of SEQ ID NO: 35 (K221E or K221D); and c) an aspartic acid or glutamicacid substitution at the position corresponding to K142 of SEQ ID NO: 35(K142E or K142D) and an aspartic acid or glutamic acid substitution atthe position corresponding to K221 of SEQ ID NO: 35 (K221E or K221D).

In certain aspects, an ALK4:ActRIIB heteromultimer of the disclosurecomprises an ActRIIB-Fc fusion protein comprising one or more amino acidmodifications that increase the pI of the ActRIIB-Fc fusion protein; andan ALK4-Fc fusion protein comprising one or more amino acidmodifications that decrease the pI of the ALK-Fc fusion protein. Forexample, an ActRIIB-Fc fusion protein may be modified by substitutingone or more neutral or negatively charged amino acids with one or morepositively charged amino acids [e.g., an arginine (R), lysine (K), orhistidine (H)]. Similarly, an ALK4-Fc fusion protein may be modified bysubstituting one or more neutral or positively charged amino acids withone or more negatively charged amino acids [e.g., aspartic acid (E) orglutamic acid (D)]. In some embodiments, the ActRIIB-Fc fusion proteinFc domain is an IgG1 Fc domain that comprises one or more amino acidmodifications that alter the pI of the ALK4-Fc fusion protein. In someembodiments, the ActRIIB-Fc fusion protein IgG1 Fc domain comprises anamino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acidsequence of SEQ ID NO: 31. In some embodiments, the ActRIIB-Fc fusionprotein IgG1 Fc domain comprises one or more amino acid substitutionsselected from: a) an amino acid substitution at the positioncorresponding to N162 of SEQ ID NO: 31; b) an amino acid substitution atthe position corresponding to D179 of SEQ ID NO: 31; and c) an aminoacid substitution at the position corresponding to N162 of SEQ ID NO: 31and an amino acid substitution at the position corresponding to D179 ofSEQ ID NO: 31. In some embodiments, the ActRIIB-Fc fusion protein IgG1Fc domain comprises one or more amino acid substitutions selected from:a) an arginine, lysine, or histidine substitution at the positioncorresponding to N162 of SEQ ID NO: 31 (N162R, N162K, or N162H); b) anarginine, lysine, or histidine substitution at the positioncorresponding to D179 of SEQ ID NO: 31 (D179R, D179K, or D179H); and c)an arginine, lysine, or histidine substitution at the positioncorresponding to N162 of SEQ ID NO: 31 (N162R, N162K, or N162H) and anarginine, lysine, or histidine substitution at the positioncorresponding to D179 of SEQ ID NO: 31 (D179R, D179K. or D179H). In someembodiments, the ActRIIB-Fc fusion protein IgG1 Fc domain comprises oneor more amino acid substitutions selected from: a) an argininesubstitution at the position corresponding to N162 of SEQ ID NO: 31(N162R); b) an arginine substitution at the position corresponding toD179 of SEQ ID NO: 31 (D179R); and c) an arginine substitution at theposition corresponding to N162 of SEQ ID NO: 31 (N162R) and an argininesubstitution at the position corresponding to D179 of SEQ ID NO: 31(D179R). In some embodiments, the ActRIIB-Fc fusion protein Fc domain isan IgG2 Fc domain that comprises one or more amino acid modificationsthat alter the pI of the ActRIIB-Fc fusion protein. In some embodiments,the ActRIIB-Fc fusion protein IgG2 Fc domain comprises an amino acidsequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more identical to the amino acid sequence of SEQID NO: 32. In some embodiments, the ActRIIB-Fc fusion protein IgG2 Fcdomain comprises one or more amino acid substitutions selected from: a)an amino acid substitution at the position corresponding to N160 of SEQID NO: 32; b) an amino acid substitution at the position correspondingto D177 of SEQ ID NO: 32; and c) an amino acid substitution at theposition corresponding to N160 of SEQ ID NO: 32 and an amino acidsubstitution at the position corresponding to D177 of SEQ ID NO: 32. Insome embodiments, the ActRIIB-Fc fusion protein IgG2 Fc domain comprisesone or more amino acid substitutions selected from: a) an arginine,lysine, or histidine substitution at the position corresponding to N160of SEQ ID NO: 32 (N160R, N160K, or N160H); b) an arginine, lysine, orhistidine substitution at the position corresponding to D177 of SEQ IDNO: 32 (D177R, D177K, or D177H); and c) an arginine, lysine, orhistidine substitution at the position corresponding to N160 of SEQ IDNO: 32 (N160R, N160K, or N160H) and an arginine, lysine, or histidinesubstitution at the position corresponding to D177 of SEQ ID NO: 32(D177R, D177K. or D177H). In some embodiments, the ActRIIB-Fc fusionprotein Fc domain is an IgG3 Fc domain that comprises one or more aminoacid modifications that alter the pI of the ActRIIB-Fc fusion protein.In some embodiments, the ActRIIB-Fc fusion protein IgG3 Fc domaincomprises an amino acid sequence that is at least 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to theamino acid sequence of SEQ ID NO: 33. In some embodiments, theActRIIB-Fc fusion protein IgG3 Fc domain comprises one or more aminoacid substitutions selected from: a) an amino acid substitution at theposition corresponding to S169 of SEQ ID NO: 33; b) an amino acidsubstitution at the position corresponding to D186 of SEQ ID NO: 33; andc) an amino acid substitution at the position corresponding to S169 ofSEQ ID NO: 33 and an amino acid substitution at the positioncorresponding to D186 of SEQ ID NO: 33. In some embodiments, themodified ActRIIB-Fc fusion protein IgG3 Fc domain comprises one or moreamino acid substitutions selected from: a) an arginine, lysine, orhistidine substitution at the position corresponding to S169 of SEQ IDNO: 33 (S169R, S169K, or S169H); b) an arginine, lysine, or histidinesubstitution at the position corresponding to D186 of SEQ ID NO: 33(D186R, D186K, or D186H); and c) an arginine, lysine, or histidinesubstitution at the position corresponding to S169 of SEQ ID NO: 33(S169R, S169K, or S169H) and an arginine, lysine, or histidinesubstitution at the position corresponding to D186 of SEQ ID NO: 33(D186R, D186K. or D186H). In some embodiments, the ActRIIB-Fc fusionprotein Fc domain is an IgG4 Fc domain that comprises one or more aminoacid modifications that alter the pI of the ActRIIB-Fc fusion protein.In some embodiments, the ActRIIB-Fc fusion protein IgG4 Fc domaincomprises an amino acid sequence that is at least 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to theamino acid sequence of SEQ ID NO: 35. In some embodiments, theActRIIB-Fc fusion protein IgG4 Fc domain comprises one or more aminoacid substitutions selected from: a) an amino acid substitution at theposition corresponding to N166 of SEQ ID NO: 35; b) an amino acidsubstitution at the position corresponding to D183 of SEQ ID NO: 35; andc) an amino acid substitution at the position corresponding to N166 ofSEQ ID NO: 35 and an amino acid substitution at position D183 of SEQ IDNO: 35. In some embodiments, the ActRIIB-Fc fusion protein IgG4 Fcdomain comprises one or more amino acid substitutions selected from: a)an arginine, lysine, or histidine substitution at the positioncorresponding to N166 of SEQ ID NO: 35 (N166R, N166K, or N166H); b) anarginine, lysine, or histidine substitution at the positioncorresponding to D183 of SEQ ID NO: 35 (D183R, D183K, or D183H); and c)an arginine, lysine, or histidine substitution at the positioncorresponding to N166 of SEQ ID NO: 35 (N166R, N166K, or N166H) and anarginine, lysine, or histidine substitution at the positioncorresponding to D183 of SEQ ID NO: 35 (D183R, D183K, or D183H). In someembodiments, the ALK4-Fc fusion protein Fc domain is an IgG1 Fc domainthat comprises one or more amino acid modifications that alter the pI ofthe ALK4-Fc fusion protein. In some embodiments, the ALK4-Fc fusionprotein IgG1 Fc domain comprises an amino acid sequence that is at least75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 31. In someembodiments, the ALK4-Fc fusion protein IgG1 fusion Fc domain comprisesone or more amino acid substitutions selected from: a) an amino acidsubstitution at the position corresponding to K138 of SEQ ID NO: 31; b)an amino acid substitution at the position corresponding to K217 of SEQID NO: 31; and c) an amino acid substitution at the positioncorresponding to K138 of SEQ ID NO: 31 and an amino acid substitution atthe position corresponding to K217 of SEQ ID NO: 31. In someembodiments, the ALK4-Fc fusion protein IgG1 Fc domain comprises one ormore amino acid substitutions selected from: a) an aspartic acid orglutamic acid substitution at the position corresponding to K138 of SEQID NO: 31 (K138E or K138D); b) an aspartic acid or glutamic acidsubstitution at the position corresponding to K217 of SEQ ID NO: 31(K217E or K217D); and c) an aspartic acid or glutamic acid substitutionat the position corresponding to K138 of SEQ ID NO: 31 (K138E or K138D)and an aspartic acid or glutamic acid substitution at the positioncorresponding to K217 of SEQ ID NO: 31 (K217E or K217D). In someembodiments, the ALK4-Fc fusion protein IgG1 Fc domain comprises one ormore amino acid substitutions selected from: a) a glutamic acidsubstitution at the position corresponding to K138 of SEQ ID NO: 31(K138E); b) an aspartic acid substitution at the position correspondingto K217 of SEQ ID NO: 31 (K217D); and c) a glutamic acid substitution atthe position corresponding to K138 of SEQ ID NO: 31 (K138E) and anaspartic acid substitution at the position corresponding to K217 of SEQID NO: 31 (K217D). In some embodiments, the ALK4-Fc fusion protein Fcdomain is an IgG2 Fc domain that comprises one or more amino acidmodifications that alter the pI of the ALK4-Fc fusion protein. In someembodiments, the ALK4-Fc fusion protein IgG2 Fc domain comprises anamino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acidsequence of SEQ ID NO: 32. In some embodiments, the ALK4-Fc fusionprotein IgG2 fusion Fc domain comprises one or more amino acidsubstitutions selected from: a) an amino acid substitution at theposition corresponding to K136 of SEQ ID NO: 32; b) an amino acidsubstitution at the position corresponding to K215 of SEQ ID NO: 32; andc) an amino acid substitution at the position corresponding to K136 ofSEQ ID NO: 32 and an amino acid substitution at the positioncorresponding to K215 of SEQ ID NO: 32. In some embodiments, the ALK4-Fcfusion protein IgG2 Fc domain comprises one or more amino acidsubstitutions selected from: a) an aspartic acid or glutamic acidsubstitution at the position corresponding to K136 of SEQ ID NO: 32(K136E or K136D); b) an aspartic acid or glutamic acid substitution atthe position corresponding to K215 of SEQ ID NO: 32 (K215E or K215D);and c) an aspartic acid or glutamic acid substitution at the positioncorresponding to K136 of SEQ ID NO: 32 (K136E or K136D) and an asparticacid or glutamic acid substitution at the position corresponding to K215of SEQ ID NO: 32 (K215E or K215D). In some embodiments, the ALK4-Fcfusion protein Fc domain is an IgG3 Fc domain that comprises one or moreamino acid modifications that alter the pI of the ALK4-Fc fusionprotein. In some embodiments, the ALK4-Fc fusion protein IgG31 Fc domaincomprises an amino acid sequence that is at least 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to theamino acid sequence of SEQ ID NO: 33. In some embodiments, the ALK4-Fcfusion protein IgG3 fusion Fc domain comprises one or more amino acidsubstitutions selected from: a) an amino acid substitution at theposition corresponding to K145 of SEQ ID NO: 33; b) an amino acidsubstitution at the position corresponding to K224 of SEQ ID NO: 33; andc) an amino acid substitution at the position corresponding to K145 ofSEQ ID NO: 33 and an amino acid substitution at the positioncorresponding to K224 of SEQ ID NO: 33. In some embodiments, the ALK4-Fcfusion protein IgG3 Fc domain comprises one or more amino acidsubstitutions selected from: a) an aspartic acid or glutamic acidsubstitution at the position corresponding to K145 of SEQ ID NO: 33(K145E or K145D); b) an aspartic acid or glutamic acid substitution atthe position corresponding to K224 of SEQ ID NO: 33 (K224E or K224D);and c) an aspartic acid or glutamic acid substitution at the positioncorresponding to K145 of SEQ ID NO: 33 (K145E or K145D) and an asparticacid or glutamic acid substitution at the position corresponding to K224of SEQ ID NO: 33 (K224E or K224D). In some embodiments, the ALK4-Fcfusion protein Fc domain is an IgG4 Fc domain that comprises one or moreamino acid modifications that alter the pI of the ALK4-Fc fusionprotein. In some embodiments, the ALK4-Fc fusion protein IgG4 Fc domaincomprises an amino acid sequence that is at least 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to theamino acid sequence of SEQ ID NO: 35. In some embodiments, the ALK4-Fcfusion protein IgG4 fusion Fc domain comprises one or more amino acidsubstitutions selected from: a) an amino acid substitution at theposition corresponding to K142 of SEQ ID NO: 35; b) an amino acidsubstitution at the position corresponding to K221 of SEQ ID NO: 35; andc) an amino acid substitution at the position corresponding to K142 ofSEQ ID NO: 35 and an amino acid substitution at the positioncorresponding to K221 of SEQ ID NO: 35. In some embodiments, the ALK4-Fcfusion protein IgG4 Fc domain comprises one or more amino acidsubstitutions selected from: a) an aspartic acid or glutamic acidsubstitution at the position corresponding to K142 of SEQ ID NO: 35(K142E or K142D); b) an aspartic acid or glutamic acid substitution atthe position corresponding to K221 of SEQ ID NO: 35 (K221E or K221D);and c) an aspartic acid or glutamic acid substitution at the positioncorresponding to K142 of SEQ ID NO: 35 (K142E or K142D) and an asparticacid or glutamic acid substitution at the position corresponding to K221of SEQ ID NO: 35 (K221E or K221D).

As described herein, ALK4-Fc fusion proteins and/or ActRIIB-Fc fusionproteins may comprise one or more modifications that promoteheteromultimer formation (e.g., ALK4-Fc:ActRIIB-Fc heterodimerization).Similarly, ALK4-Fc fusion proteins and/or ActRIIB-Fc fusion proteins maycomprise one or more modifications that inhibit homomultimer formation(e.g., ALK4-Fc:ActRIIB-Fc homodimerization). In some embodiments,ALK4-Fc fusion proteins and/or ActRIIB-Fc fusion proteins may compriseone or more modifications that promote heteromultimer formation andcomprise one or more modifications that inhibit homomultimer formation.For example, in some embodiments, an ALK4:ActRIIB heteromultimercomprises: a) an ALK4-Fc fusion protein having an IgG1 Fc domaincomprising a cysteine substitution at position S132 of SEQ ID NO: 31(S132C) and a tryptophan substitution at position T144 of SEQ ID NO: 31(T144W); and b) an ActRIIB-Fc fusion protein having an IgG1 Fc domaincomprising a cysteine substitution at position Y127 of SEQ ID NO: 31(Y127C), a serine substitution at position T144 of SEQ ID NO: 31(T144S), an alanine substitution at position L146 of SEQ ID NO: 31(L146A), and a valine substitution at position Y185 of SEQ ID NO: 31(Y185V). In some embodiments, an ALK4:ActRIIB heteromultimer comprises:a) an ActRIIB-Fc fusion protein having an IgG1 Fc domain comprising acysteine substitution at position S132 of SEQ ID NO: 31 (S132C) and atryptophan substitution at position T144 of SEQ ID NO: 31 (T144W); andb) an ALK4-Fc fusion protein having an IgG1 Fc domain comprising acysteine substitution at position Y127 of SEQ ID NO: 31 (Y127C), aserine substitution at position T144 of SEQ ID NO: 31 (T144S), analanine substitution at position L146 of SEQ ID NO: 31 (L146A), and avaline substitution at position Y185 of SEQ ID NO: 31 (Y185V). In someembodiments, an ALK4:ActRIIB heteromultimer comprises: a) an ALK4-Fcfusion protein having an IgG2 Fc domain comprising a cysteinesubstitution at position S130 of SEQ ID NO: 32 (S130C) and a tryptophansubstitution at position T142 of SEQ ID NO: 32 (T142W); and b) anActRIIB-Fc fusion protein having an IgG2 Fc domain comprising a cysteinesubstitution at position Y125 of SEQ ID NO: 32 (Y125C), a serinesubstitution at position T142 of SEQ ID NO: 32 (T142S), an alaninesubstitution at position L144 of SEQ ID NO: 32 (L144A), and a valinesubstitution at position Y183 of SEQ ID NO: 32 (Y183V). In someembodiments, an ALK4:ActRIIB heteromultimer comprises: a) an ActRIIB-Fcfusion protein having an IgG2 Fc domain comprising a cysteinesubstitution at position S130 of SEQ ID NO: 32 (S130C) and a tryptophansubstitution at position T142 of SEQ ID NO: 32 (T142W); and b) anALK4-Fc fusion protein having an IgG2 Fc domain comprising a cysteinesubstitution at position Y125 of SEQ ID NO: 32 (Y125C), a serinesubstitution at position T142 of SEQ ID NO: 32 (T142S), an alaninesubstitution at position L144 of SEQ ID NO: 32 (L144A), and a valinesubstitution at position Y183 of SEQ ID NO: 32 (Y183V). In someembodiments, an ALK4:ActRIIB heteromultimer comprises: a) an ALK4-Fcfusion protein having an IgG3 Fc domain comprising a cysteinesubstitution at position S139 of SEQ ID NO: 33 (S139C) and a tryptophansubstitution at position T151 of SEQ ID NO: 33 (T151W); and b) theActRIIB-Fc fusion protein having an IgG3 Fc domain comprising a cysteinesubstitution at position Y134 of SEQ ID NO: 33 (Y134C), a serinesubstitution at position T151 of SEQ ID NO: 33 (T151S), an alaninesubstitution at position L153 of SEQ ID NO: 33 (L153A), and a valinesubstitution at position Y192 of SEQ ID NO: 33 (Y192V). In someembodiments, an ALK4:ActRIIB heteromultimer comprises: a) an ActRIIB-Fcfusion protein having an IgG3 Fc domain comprising a cysteinesubstitution at position S139 of SEQ ID NO: 33 (S139C) and a tryptophansubstitution at position T151 of SEQ ID NO: 33 (T151W); and b) anALK4-Fc fusion protein having an IgG3 Fc domain comprising a cysteinesubstitution at position Y134 of SEQ ID NO: 33 (Y134C), a serinesubstitution at position T151 of SEQ ID NO: 33 (T151S), an alaninesubstitution at position L153 of SEQ ID NO: 33 (L153A), and a valinesubstitution at position Y192 of SEQ ID NO: 33 (Y192V). In someembodiments, an ALK4:ActRIIB heteromultimer comprises: a) an ALK4-Fcfusion protein having an IgG4 Fc domain comprises a cysteinesubstitution at position 5136 of SEQ ID NO: 35 (S136C) and a tryptophansubstitution at position T148 of SEQ ID NO: 35 (T148W); and b) anActRIIB-Fc fusion protein having an IgG4 Fc domain comprises a cysteinesubstitution at position Y131 of SEQ ID NO: 35 (Y131C), a serinesubstitution at position T148 of SEQ ID NO: 35 (T148S), an alaninesubstitution at position L150 of SEQ ID NO: 35 (L150A), and a valinesubstitution at position Y189 of SEQ ID NO: 35 (Y189V). In someembodiments, an ALK4:ActRIIB heteromultimer comprises: a) an ActRIIB-Fcfusion protein having an IgG4 Fc domain comprising a cysteinesubstitution at position S136 of SEQ ID NO: 35 (S136C) and a tryptophansubstitution at position T148 of SEQ ID NO: 35 (T148W); and b) anALK4-Fc fusion protein having an IgG4 Fc domain comprising a cysteinesubstitution at position Y131 of SEQ ID NO: 35 (Y131C), a serinesubstitution at position T148 of SEQ ID NO: 35 (T148S), an alaninesubstitution at position L150 of SEQ ID NO: 35 (L150A), and a valinesubstitution at position Y189 of SEQ ID NO: 35 (Y189V).

In certain aspects, an ALK4:ActRIIB heteromultimer of the disclosurecomprises: a) an ALK4-Fc fusion protein having an Fc domain that is atleast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% identical to the amino acid sequence of SEQ ID NO: 66; and b) anActRIIB-Fc fusion protein having an Fc domain that is at least 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto the amino acid sequence of SEQ ID NO: 67. In some embodiments, theALK4-Fc fusion protein Fc domain comprises one or more amino acidsubstitutions selected from: a) a glutamic acid at the positioncorresponding to 138 of SEQ ID NO: 66; b) an aspartic acid at theposition corresponding to 217 of SEQ ID NO: 66; and c) a glutamic acidat the position corresponding to 138 of SEQ ID NO: 66 and an asparticacid at the position corresponding to 217 of SEQ ID NO: 66. Optionally,the ALK4-Fc fusion protein Fc domain further comprises a cysteine at theposition corresponding to 132 of SEQ ID NO: 66 and a tryptophan at theposition corresponding to 144 of SEQ ID NO: 66. In some embodiments, theActRIIB-Fc fusion protein Fc domain comprises one or more amino acidsubstitutions selected from: a) an arginine at the positioncorresponding to 162 of SEQ ID NO: 67; b) an arginine at the positioncorresponding to 179 of SEQ ID NO: 67; and c) an arginine at theposition corresponding to 162 of SEQ ID NO: 67 and an arginine at theposition corresponding to 179 of SEQ ID NO: 67. Optionally, theActRIIB-Fc fusion protein Fc domain further comprises a cysteine at theposition corresponding to 127 of SEQ ID NO: 67, a serine at the positioncorresponding to 144 of SEQ ID NO: 67, an alanine at the positioncorresponding to 146 of SEQ ID NO: 67, and a valine at the positioncorresponding to 185 of SEQ ID NO: 67.

In certain aspects, an ALK4:ActRIIB heteromultimer of the disclosurecomprises: a) an ActRIIB-Fc fusion protein having an Fc domain that isat least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to the amino acid sequence of SEQ ID NO: 66; andb) an ALK4-Fc fusion protein having an Fc domain that is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the amino acid sequence of SEQ ID NO: 67. In someembodiments, the ActRIIB-Fc fusion protein Fc domain comprises one ormore amino acid substitutions selected from: a) a glutamic acid at theposition corresponding to 138 of SEQ ID NO: 66; b) an aspartic acid atthe position corresponding to 217 of SEQ ID NO: 66; and c) a glutamicacid at the position corresponding to 138 of SEQ ID NO: 66 and anaspartic acid at the position corresponding to 217 of SEQ ID NO: 66.Optionally, the ActRIIB-Fc fusion protein Fc domain further comprises acysteine at the position corresponding to 132 of SEQ ID NO: 66 and atryptophan at the position corresponding to 144 of SEQ ID NO: 66. Insome embodiments, the ALK4-Fc fusion protein Fc domain comprises one ormore amino acid substitutions selected from: a) an arginine at theposition corresponding to 162 of SEQ ID NO: 67; b) an arginine at theposition corresponding to 179 of SEQ ID NO: 67; and c) an arginine atthe position corresponding to 162 of SEQ ID NO: 67 and an arginine atthe position corresponding to 179 of SEQ ID NO: 67. Optionally, theALK4-Fc fusion protein Fc domain further comprises a cysteine at theposition corresponding to 127 of SEQ ID NO: 67, a serine at the positioncorresponding to 144 of SEQ ID NO: 67, an alanine at the positioncorresponding to 146 of SEQ ID NO: 67, and a valine at the positioncorresponding to 185 of SEQ ID NO: 67.

In certain aspects, the disclosure relates to a recombinant ALK4:ActRIIBheteromultimer comprising at least one ALK4-Fc fusion protein and atleast one ActRIIB-Fc fusion protein, wherein: a) the ALK4-Fc fusionprotein comprises an IgG1 Fc domain comprising a cysteine at theposition corresponding to S132 of SEQ ID NO: 31 (S132C), a tryptophan atthe position corresponding to T144 of SEQ ID NO: 31 (T144W), and anacidic amino acid at the position corresponding to H213 of SEQ ID NO:31; and b) the ActRIIB-Fc fusion protein comprises an IgG1 Fc domaincomprising a cysteine at the position corresponding to Y127 of SEQ IDNO: 31 (Y127C), a serine at the position corresponding to T144 of SEQ IDNO: 31 (T144S), an alanine at the position corresponding to L146 of SEQID NO: 31 (L146A), and a valine at the position corresponding to Y185 ofSEQ ID NO: 31 (Y185V). In some embodiments, wherein the acidic aminoacid at the position corresponding to H213 of SEQ ID NO: 31 is anaspartic acid. In some embodiments, the acidic amino acid at theposition corresponding to H213 of SEQ ID NO: 31 is a glutamic acid. Insome embodiments, the ALK4-Fc fusion protein Fc domain is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 31. In someembodiments, the ActRIIB-Fc fusion protein Fc domain is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 31.

In certain aspects, the disclosure relates to a recombinant ALK4:ActRIIBheteromultimer comprising at least one ALK4-Fc fusion protein and atleast one ActRIIB-Fc fusion protein, wherein: a) the ActRIIB-Fc fusionprotein comprises an IgG1 Fc domain comprising a cysteine at theposition corresponding to S132 of SEQ ID NO: 31 (S132C), a tryptophan atthe position corresponding to T144 of SEQ ID NO: 31 (T144W), and anacidic amino acid at the position corresponding to H213 of SEQ ID NO:31; and b) the ALK4-Fc fusion protein comprises an IgG1 Fc domaincomprising a cysteine at the position corresponding to Y127 of SEQ IDNO: 31 (Y127C), a serine at the position corresponding to T144 of SEQ IDNO: 31 (T144S), an alanine at the position corresponding to L146 of SEQID NO: 31 (L146A), and a valine at the position corresponding to Y185 ofSEQ ID NO: 31 (Y185V). In some embodiments, wherein the acidic aminoacid at the position corresponding to H213 of SEQ ID NO: 31 is anaspartic acid. In some embodiments, the acidic amino acid at theposition corresponding to H213 of SEQ ID NO: 31 is a glutamic acid. Insome embodiments, the ALK4-Fc fusion protein Fc domain is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 31. In someembodiments, the ActRIIB-Fc fusion protein Fc domain is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 31. In certainaspects, the disclosure relates to a recombinant ALK4:ActRIIBheteromultimer comprising at least one ALK4-Fc fusion protein and atleast one ActRIIB-Fc fusion protein, wherein: a) the ALK4-Fc fusionprotein comprises an IgG1 Fc domain comprising a cysteine at theposition corresponding to S132 of SEQ ID NO: 31 (S132C), and atryptophan at the position corresponding to T144 of SEQ ID NO: 31(T144W); and b) the ActRIIB-Fc fusion protein comprises an IgG1 Fcdomain comprising a cysteine at the position corresponding to Y127 ofSEQ ID NO: 31 (Y127C), a serine at the position corresponding to T144 ofSEQ ID NO: 31 (T144S), an alanine at the position corresponding to L146of SEQ ID NO: 31 (L146A), a valine at the position corresponding to Y185of SEQ ID NO: 31 (Y185V), and an acidic amino acid at the positioncorresponding to H213 of SEQ ID NO: 31. In some embodiments, wherein theacidic amino acid at the position corresponding to H213 of SEQ ID NO: 31is an aspartic acid. In some embodiments, the acidic amino acid at theposition corresponding to H213 of SEQ ID NO: 31 is a glutamic acid. Insome embodiments, the ALK4-Fc fusion protein Fc domain is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 31. In someembodiments, the ActRIIB-Fc fusion protein Fc domain is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 31. In certainaspects, the disclosure relates to a recombinant ALK4:ActRIIBheteromultimer comprising at least one ALK4-Fc fusion protein and atleast one ActRIIB-Fc fusion protein, wherein: a) the ActRIIB-Fc fusionprotein comprises an IgG1 Fc domain comprising a cysteine at theposition corresponding to S132 of SEQ ID NO: 31 (S132C), and atryptophan at the position corresponding to T144 of SEQ ID NO: 31(T144W); and b) the ALK4-Fc fusion protein comprises an IgG1 Fc domaincomprising a cysteine at the position corresponding to Y127 of SEQ IDNO: 31 (Y127C), a serine at the position corresponding to T144 of SEQ IDNO: 31 (T144S), an alanine at the position corresponding to L146 of SEQID NO: 31 (L146A), and a valine at the position corresponding to Y185 ofSEQ ID NO: 31 (Y185V), and an acidic amino acid at the positioncorresponding to H213 of SEQ ID NO: 31. In some embodiments, wherein theacidic amino acid at the position corresponding to H213 of SEQ ID NO: 31is an aspartic acid. In some embodiments, the acidic amino acid at theposition corresponding to H213 of SEQ ID NO: 31 is a glutamic acid. Insome embodiments, the ALK4-Fc fusion protein Fc domain is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 31. In someembodiments, the ActRIIB-Fc fusion protein Fc domain is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 31.

In certain aspects, the disclosure relates to a recombinant ALK4:ActRIIBheteromultimer comprising at least one ALK4-Fc fusion protein and atleast one ActRIIB-Fc fusion protein, wherein: a) the ALK4-Fc fusionprotein comprises an IgG2 Fc domain comprising a cysteine at theposition corresponding to S130 of SEQ ID NO: 32 (S130C), a tryptophan atthe position corresponding to T142 of SEQ ID NO: 32 (T142W), and anacidic amino acid at the position corresponding to H211 of SEQ ID NO:32; and b) the ActRIIB-Fc fusion protein comprises an IgG2 Fc domaincomprising a cysteine at the position corresponding to Y125 of SEQ IDNO: 32 (Y125C), a serine at the position corresponding to T142 of SEQ IDNO: 32 (T142S), an alanine at the position corresponding to L144 of SEQID NO: 32 (L144A), and a valine at the position corresponding to Y183 ofSEQ ID NO: 32 (Y183\). In some embodiments, wherein the acidic aminoacid at the position corresponding to H211 of SEQ ID NO: 32 is anaspartic acid. In some embodiments, the acidic amino acid at theposition corresponding to H211 of SEQ ID NO: 32 is a glutamic acid. Insome embodiments, the ALK4-Fc fusion protein Fc domain is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 32. In someembodiments, the ActRIIB-Fc fusion protein Fc domain is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 32.

In certain aspects, the disclosure relates to a recombinant ALK4:ActRIIBheteromultimer comprising at least one ALK4-Fc fusion protein and atleast one ActRIIB-Fc fusion protein, wherein: a) the ActRIIB-Fc fusionprotein comprises an IgG2 Fc domain comprising a cysteine at theposition corresponding to S130 of SEQ ID NO: 32 (S130C), a tryptophan atthe position corresponding to T142 of SEQ ID NO: 32 (T142W), and anacidic amino acid at the position corresponding to H211 of SEQ ID NO:32; and b) the ALK4-Fc fusion protein comprises an IgG2 Fc domaincomprising a cysteine at the position corresponding to Y125 of SEQ IDNO: 32 (Y125C), a serine at the position corresponding to T142 of SEQ IDNO: 32 (T142S), an alanine at the position corresponding to L144 of SEQID NO: 32 (L144A), and a valine at the position corresponding to Y183 ofSEQ ID NO: 32 (Y183V). In some embodiments, wherein the acidic aminoacid at the position corresponding to H211 of SEQ ID NO: 32 is anaspartic acid. In some embodiments, the acidic amino acid at theposition corresponding to H211 of SEQ ID NO: 32 is a glutamic acid. Insome embodiments, the ALK4-Fc fusion protein Fc domain is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 32. In someembodiments, the ActRIIB-Fc fusion protein Fc domain is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 32.

In certain aspects, the disclosure relates to a recombinant ALK4:ActRIIBheteromultimer comprising at least one ALK4-Fc fusion protein and atleast one ActRIIB-Fc fusion protein, wherein: a) the ALK4-Fc fusionprotein comprises an IgG2 Fc domain comprising a cysteine at theposition corresponding to S130 of SEQ ID NO: 32 (S130C), and atryptophan at the position corresponding to T142 of SEQ ID NO: 32(T142W); and b) the ActRIIB-Fc fusion protein comprises an IgG2 Fcdomain comprising a cysteine at the position corresponding to Y125 ofSEQ ID NO: 32 (Y125C), a serine at the position corresponding to T142 ofSEQ ID NO: 32 (T142S), an alanine at the position corresponding to L144of SEQ ID NO: 32 (L144A), a valine at the position corresponding to Y183of SEQ ID NO: 32 (Y183V), and an acidic amino acid at the positioncorresponding to H211 of SEQ ID NO: 32. In some embodiments, wherein theacidic amino acid at the position corresponding to H211 of SEQ ID NO: 32is an aspartic acid. In some embodiments, the acidic amino acid at theposition corresponding to H211 of SEQ ID NO: 32 is a glutamic acid. Insome embodiments, the ALK4-Fc fusion protein Fc domain is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 32. In someembodiments, the ActRIIB-Fc fusion protein Fc domain is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 32.

In certain aspects, the disclosure relates to a recombinant ALK4:ActRIIBheteromultimer comprising at least one ALK4-Fc fusion protein and atleast one ActRIIB-Fc fusion protein, wherein: a) the ActRIIB-Fc fusionprotein comprises an IgG2 Fc domain comprising a cysteine at theposition corresponding to S130 of SEQ ID NO: 32 (S1300, and a tryptophanat the position corresponding to T142 of SEQ ID NO: 32 (T142W); and b)the ALK4-Fc fusion protein comprises an IgG2 Fc domain comprising acysteine at the position corresponding to Y125 of SEQ ID NO: 32 (Y125C),a serine at the position corresponding to T142 of SEQ ID NO: 32 (T142S),an alanine at the position corresponding to L144 of SEQ ID NO: 32(L144A), a valine at the position corresponding to Y183 of SEQ ID NO: 32(Y183\), and an acidic amino acid at the position corresponding to H211of SEQ ID NO: 32. In some embodiments, wherein the acidic amino acid atthe position corresponding to H211 of SEQ ID NO: 32 is an aspartic acid.In some embodiments, the acidic amino acid at the position correspondingto H211 of SEQ ID NO: 32 is a glutamic acid. In some embodiments, theALK4-Fc fusion protein Fc domain is at least 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the aminoacid sequence of SEQ ID NO: 32. In some embodiments, the ActRIIB-Fcfusion protein Fc domain is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more identical to the amino acidsequence of SEQ ID NO: 32.

In certain aspects, the disclosure relates to a recombinant ALK4:ActRIIBheteromultimer comprising at least one ALK4-Fc fusion protein and atleast one ActRIIB-Fc fusion protein, wherein: a) the ALK4-Fc fusionprotein comprises an IgG4 Fc domain comprising a cysteine at theposition corresponding to S136 of SEQ ID NO: 35 (S136C), a tryptophan atthe position corresponding to T148 of SEQ ID NO: 35 (T148W), and anacidic amino acid at the position corresponding to H217 of SEQ ID NO:35; and b) the ActRIIB-Fc fusion protein comprises an IgG4 Fc domaincomprising a cysteine at the position corresponding to Y131 of SEQ IDNO: 35 (Y131C), a serine at the position corresponding to T148 of SEQ IDNO: 35 (T148S), an alanine at the position corresponding to L150 of SEQID NO: 35 (L150A), and a valine at the position corresponding to Y189 ofSEQ ID NO: 35 (Y189V). In some embodiments, wherein the acidic aminoacid at the position corresponding to H217 of SEQ ID NO: 35 is anaspartic acid. In some embodiments, the acidic amino acid at theposition corresponding to H217 of SEQ ID NO: 35 is a glutamic acid. Insome embodiments, the ALK4-Fc fusion protein Fc domain is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 35. In someembodiments, the ActRIIB-Fc fusion protein Fc domain is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 35.

In certain aspects, the disclosure relates to a recombinant ALK4:ActRIIBheteromultimer comprising at least one ALK4-Fc fusion protein and atleast one ActRIIB-Fc fusion protein, wherein: a) the ActRIIB-Fc fusionprotein comprises an IgG4 Fc domain comprising a cysteine at theposition corresponding to S136 of SEQ ID NO: 35 (S136C), a tryptophan atthe position corresponding to T148 of SEQ ID NO: 35 (T148W), and anacidic amino acid at the position corresponding to H217 of SEQ ID NO:35; and b) the ALK4-Fc fusion protein comprises an IgG4 Fc domaincomprising a cysteine at the position corresponding to Y131 of SEQ IDNO: 35 (Y131C), a serine at the position corresponding to T148 of SEQ IDNO: 35 (T148S), an alanine at the position corresponding to L150 of SEQID NO: 35 (L150A), and a valine at the position corresponding to Y189 ofSEQ ID NO: 35 (Y189V). In some embodiments, wherein the acidic aminoacid at the position corresponding to H217 of SEQ ID NO: 35 is anaspartic acid. In some embodiments, the acidic amino acid at theposition corresponding to H217 of SEQ ID NO: 35 is a glutamic acid. Insome embodiments, the ALK4-Fc fusion protein Fc domain is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 35. In someembodiments, the ActRIIB-Fc fusion protein Fc domain is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 35.

In certain aspects, the disclosure relates to recombinant ALK4:ActRIIBheteromultimer comprising at least one ALK4-Fc fusion protein and atleast one ActRIIB-Fc fusion protein, wherein: a) the ALK4-Fc fusionprotein comprises an IgG4 Fc domain comprising a cysteine at theposition corresponding to S136 of SEQ ID NO: 35 (S136C), and atryptophan at the position corresponding to T148 of SEQ ID NO: 35(T148W); and b) the ActRIIB-Fc fusion protein comprises an IgG4 Fcdomain comprising a cysteine at the position corresponding to Y131 ofSEQ ID NO: 35 (Y131C), a serine at the position corresponding to T148 ofSEQ ID NO: 35 (T148S), an alanine at the position corresponding to L150of SEQ ID NO: 35 (L150A), a valine at the position corresponding to Y189of SEQ ID NO: 35 (Y189V), and an acidic amino acid at the positioncorresponding to H217 of SEQ ID NO: 35. In some embodiments, wherein theacidic amino acid at the position corresponding to H217 of SEQ ID NO: 35is an aspartic acid. In some embodiments, the acidic amino acid at theposition corresponding to H217 of SEQ ID NO: 35 is a glutamic acid. Insome embodiments, the ALK4-Fc fusion protein Fc domain is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 35. In someembodiments, the ActRIIB-Fc fusion protein Fc domain is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 35.

In certain aspects, the disclosure relates to recombinant ALK4:ActRIIBheteromultimer comprising at least one ALK4-Fc fusion protein and atleast one ActRIIB-Fc fusion protein, wherein: a) the ActRIIB-Fc fusionprotein comprises an IgG4 Fc domain comprising a cysteine at theposition corresponding to S136 of SEQ ID NO: 35 (S136C), and atryptophan at the position corresponding to T148 of SEQ ID NO: 35(T148W); and b) the ALK4-Fc fusion protein comprises an IgG4 Fc domaincomprising a cysteine at the position corresponding to Y131 of SEQ IDNO: 35 (Y131C), a serine at the position corresponding to T148 of SEQ IDNO: 35 (T148S), an alanine at the position corresponding to L150 of SEQID NO: 35 (L150A), a valine at the position corresponding to Y189 of SEQID NO: 35 (Y189V), and an acidic amino acid at the positioncorresponding to H217 of SEQ ID NO: 35. In some embodiments, wherein theacidic amino acid at the position corresponding to H217 of SEQ ID NO: 35is an aspartic acid. In some embodiments, the acidic amino acid at theposition corresponding to H217 of SEQ ID NO: 35 is a glutamic acid. Insome embodiments, the ALK4-Fc fusion protein Fc domain is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 35. In someembodiments, the ActRIIB-Fc fusion protein Fc domain is at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to the amino acid sequence of SEQ ID NO: 35.

Optionally, ALK4 polypeptides are connected directly (fused) to one ormore heterologous domains, or an intervening sequence, such as a linker,may be positioned between the amino acid sequence of the ALK4polypeptide and the one or more heterologous domains (e.g., an Fc domainof an immunoglobulin). Similarly, the ActRIIB polypeptide may beconnected directly (fused) to one or more heterologous domains, or anintervening sequence, such as a linker, may be positioned between theamino acid sequence of the ActRIIB polypeptide and the one or moreheterologous domains (e.g., an Fc domain of an immunoglobulin). Linkersmay correspond to the roughly 15 amino acid unstructured region at theC-terminal end of the extracellular domain of ActRIIB or ALK4 (the“tail”), or it may be an artificial sequence of between 5 and 15, 20,30, 50, 100 or more amino acids that are relatively free of secondarystructure. A linker may be rich in glycine and proline residues and may,for example, contain repeating sequences of threonine/serine andglycines.

Examples of linkers include, but are not limited to, the sequences TGGG(SEQ ID NO: 17), SGGG (SEQ ID NO: 18), TGGGG (SEQ ID NO: 15), SGGGG (SEQID NO: 16), GGGGS (SEQ ID NO: 58), GGGG (SEQ ID NO: 14), and GGG (SEQ IDNO: 13).

In certain aspects, the disclosure relates to a recombinant ALK4:ActRIIBheteromultimer comprising at least one ALK4-Fc fusion protein and atleast one ActRIIB-Fc fusion protein, wherein the ALK4-Fc fusion proteincomprises an amino acid sequence that is at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% to the amino acid sequence of SEQ ID NO: 76, and wherein theActRIIB-Fc fusion protein comprises an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% to the amino acid sequence of SEQ IDNO: 72. In some embodiments, the ALK4-Fc fusion protein comprises one ormore amino acid selected from: a) a cysteine at the positioncorresponding to 234 of SEQ ID NO: 76, a serine at the positioncorresponding to 251 of SEQ ID NO: 76, an alanine at the positioncorresponding to 253 of SEQ ID NO: 76, and a valine at the positioncorresponding to 292 of SEQ ID NO: 76; b) a positively charged aminoacid at the position corresponding to 269 of SEQ ID NO: 76; c) apositively charged amino acid at the position corresponding to D286 ofSEQ ID NO: 76; d) a positively charged amino acid at the positioncorresponding to 269 of SEQ ID NO: 76 and a positively charged aminoacid at the position corresponding to 286 of SEQ ID NO: 76; e) acysteine at the position corresponding to 234 of SEQ ID NO: 76, a serineat the position corresponding to 251 of SEQ ID NO: 76, an alanine at theposition corresponding to 253 of SEQ ID NO: 76, a valine at the positioncorresponding to 292 of SEQ ID NO: 76 (Y292V), and a positively chargedamino acid at the position corresponding to 269 of SEQ ID NO: 76; f) acysteine at the position corresponding to 234 of SEQ ID NO: 76, a serineat the position corresponding to 251 of SEQ ID NO: 76, an alanine at theposition corresponding to 253 of SEQ ID NO: 76, a valine at position 292of SEQ ID NO: 76, and a positively charged amino acid at the positioncorresponding to 286 of SEQ ID NO: 76; and g) a cysteine at the positioncorresponding to 234 of SEQ ID NO: 76, a serine at the positioncorresponding to 251 of SEQ ID NO: 76, an alanine at the positioncorresponding to 253 of SEQ ID NO: 76, and a valine at the positioncorresponding to 292 of SEQ ID NO: 76, a positively charged amino acidat the position corresponding to 269 of SEQ ID NO: 76, and a positivelycharged amino acid at the position corresponding to 286 of SEQ ID NO:76. In some embodiments, the ActRIIB-Fc fusion protein comprises one ormore amino acid selected from: a) a cysteine at the positioncorresponding to 250 of SEQ ID NO: 72, and a tryptophan at position 262of SEQ ID NO: 72; b) a negatively charged amino acid at the positioncorresponding to 256 of SEQ ID NO: 72; c) a negatively charged aminoacid at the position corresponding to 335 of SEQ ID NO: 72; d) anegatively charged amino acid at the position corresponding to 256 ofSEQ ID NO: 72 and a negatively charged amino acid at the positioncorresponding to 335 of SEQ ID NO: 72; e) a cysteine at the positioncorresponding to 250 of SEQ ID NO: 72, a tryptophan at position 262 ofSEQ ID NO: 72, and a negatively charged amino acid at the positioncorresponding to 256 of SEQ ID NO: 72; f) a cysteine at the positioncorresponding to 250 of SEQ ID NO: 72, a tryptophan at position 262 ofSEQ ID NO: 72, and a negatively charged amino acid at the positioncorresponding to 335 of SEQ ID NO: 72; and g) a cysteine at the positioncorresponding to 250 of SEQ ID NO: 72, a tryptophan at position 262 ofSEQ ID NO: 72, a negatively charged amino acid at the positioncorresponding to 256 of SEQ ID NO: 72, and a negatively charged aminoacid at the position corresponding to 335 of SEQ ID NO: 72. In someembodiments, the positively charged amino acid residue is a modified ornaturally occurring (e.g., R, K, or H) amino acid. In some embodiments,the positively charged amino acid residue is R. In some embodiments, thenegatively charged amino acid residue is a modified or naturallyoccurring (e.g., D or E) amino acid. In some embodiments, the negativelycharged amino acid residue is D. In some embodiments, the negativelycharged amino acid residue is E.

In certain aspects, the disclosure relates to a recombinant ALK4-Fcfusion protein comprises an amino acid sequence that is at least 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% to the amino acid sequence of SEQ ID NO: 76. Insome embodiments, the ALK4-Fc fusion protein Fc domain comprises one ormore amino acid selected from: a) a cysteine at the positioncorresponding to 234 of SEQ ID NO: 76, a serine at the positioncorresponding to 251 of SEQ ID NO: 76, an alanine at the positioncorresponding to 253 of SEQ ID NO: 76, and a valine at the positioncorresponding to 292 of SEQ ID NO: 76; b) a positively charged aminoacid at the position corresponding to 269 of SEQ ID NO: 76; c) apositively charged amino acid at the position corresponding to 286 ofSEQ ID NO: 76; d) a positively charged amino acid at the positioncorresponding to 269 of SEQ ID NO: 76 and a positively charged aminoacid at the position corresponding to 286 of SEQ ID NO: 76; e) acysteine at the position corresponding to 234 of SEQ ID NO: 76, a serineat the position corresponding to 251 of SEQ ID NO: 76, an alanine at theposition corresponding to 253 of SEQ ID NO: 76, a valine at position 292of SEQ ID NO: 76, and a positively charged amino acid at the positioncorresponding to 269 of SEQ ID NO: 76; f) a cysteine at the positioncorresponding to 234 of SEQ ID NO: 76, a serine at the positioncorresponding to 251 of SEQ ID NO: 76, an alanine at the positioncorresponding to 253 of SEQ ID NO: 76, a valine at the positioncorresponding to 292 of SEQ ID NO: 76, and a positively charged aminoacid at the position corresponding to 286 of SEQ ID NO: 76; and g) acysteine at the position corresponding to 234 of SEQ ID NO: 76, a serineat the position corresponding to 251 of SEQ ID NO: 76, an alanine at theposition corresponding to 253 of SEQ ID NO: 76, and a valine at theposition corresponding to 292 of SEQ ID NO: 76, a positively chargedamino acid at the position corresponding to 269 of SEQ ID NO: 76, and apositively charged amino acid at the position corresponding to 286 ofSEQ ID NO: 76. In some embodiments, the positively charged amino acidresidue is a modified or naturally occurring (e.g., R, K, or H) aminoacid. In some embodiments, the positively charged amino acid residue isR.

In certain aspects, the disclosure relates to a recombinant ActRIIB-Fcfusion protein comprises an amino acid sequence that is at least 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% to the amino acid sequence of SEQ ID NO: 72. Insome embodiments, the fusion protein comprises one or more amino acidselected from: a) a cysteine at the position corresponding to 250 of SEQID NO: 72 and a tryptophan at the position corresponding to 262 of SEQID NO: 72; b) a negatively charged amino acid at the positioncorresponding to 256 of SEQ ID NO: 72; c) a negatively charged aminoacid at the position corresponding to 335 of SEQ ID NO: 72; d) anegatively charged amino acid at the position corresponding to 256 ofSEQ ID NO: 72 and a negatively charged amino acid at the positioncorresponding to 335 of SEQ ID NO: 72; e) a cysteine at the positioncorresponding to 250 of SEQ ID NO: 72, a tryptophan at position 262 ofSEQ ID NO: 72, and a negatively charged amino acid at the positioncorresponding to 256 of SEQ ID NO: 72; f) a cysteine at the positioncorresponding to 250 of SEQ ID NO: 72, a tryptophan at position 262 ofSEQ ID NO: 72, and a negatively charged amino acid at the positioncorresponding to 335 of SEQ ID NO: 72; and g) a cysteine at the positioncorresponding to 250 of SEQ ID NO: 7, a tryptophan at position 262 ofSEQ ID NO: 72, a negatively charged amino acid at the positioncorresponding to 256 of SEQ ID NO: 72, and a negatively charged aminoacid at the position corresponding to 335 of SEQ ID NO: 72. In someembodiments, the negatively charged amino acid residue is a modified ornaturally occurring (e.g., D or E) amino acid. In some embodiments, thenegatively charged amino acid residue is D. In some embodiments, thenegatively charged amino acid residue is E.

In certain aspects, the disclosure relates to a recombinant ALK4-Fcfusion protein comprises an amino acid sequence that is at least 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% to the amino acid sequence of SEQ ID NO: 74. Insome embodiments, the ALK4-Fc fusion protein Fc domain comprises one ormore amino acid selected from: a) a cysteine at the positioncorresponding to 258 of SEQ ID NO: 74, a serine at the positioncorresponding to 275 of SEQ ID NO: 74, an alanine at the positioncorresponding to 277 of SEQ ID NO: 74, and a valine at position 316 ofSEQ ID NO: 74; b) a positively charged amino acid at the positioncorresponding to 293 of SEQ ID NO: 74; c) a positively charged aminoacid at the position corresponding to 310 of SEQ ID NO: 74; d) apositively charged amino acid at the position corresponding to 293 ofSEQ ID NO: 74 and a positively charged amino acid at the positioncorresponding to 310 of SEQ ID NO: 74; e) a cysteine at the positioncorresponding to 258 of SEQ ID NO: 74, a serine at the positioncorresponding to 275 of SEQ ID NO: 74, an alanine at the positioncorresponding to 277 of SEQ ID NO: 74, a valine at position 316 of SEQID NO: 74, and a positively charged amino acid at the positioncorresponding to 293 of SEQ ID NO: 74; f) a cysteine at the positioncorresponding to 258 of SEQ ID NO: 74, a serine at the positioncorresponding to 275 of SEQ ID NO: 74, an alanine at the positioncorresponding to 277 of SEQ ID NO: 74, a valine at the positioncorresponding to 316 of SEQ ID NO: 74, and a positively charged aminoacid at the position corresponding to 310 of SEQ ID NO: 74 g) a cysteineat the position corresponding to 258 of SEQ ID NO: 74, a serine at theposition corresponding to 275 of SEQ ID NO: 74, an alanine at theposition corresponding to 277 of SEQ ID NO: 74, a valine at the positioncorresponding to 316 of SEQ ID NO: 74, a positively charged amino acidat the position corresponding to 293 of SEQ ID NO: 74, and a positivelycharged amino acid at the position corresponding to 310 of SEQ ID NO:74. In some embodiments, the positively charged amino acid residue is amodified or naturally occurring (e.g., R, K, or H) amino acid. In someembodiments, the positively charged amino acid residue is R.

In certain aspects, the disclosure relates to a recombinant ActRIIB-Fcfusion protein comprises an amino acid sequence that is at least 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% to the amino acid sequence of SEQ ID NO: 70. Insome embodiments, the ActRIIB-Fc fusion protein Fc domain comprises oneor more amino acid selected from: a) a cysteine at the positioncorresponding to 275 of SEQ ID NO: 70 and a tryptophan at the positioncorresponding to 287 of SEQ ID NO: 70; b) a negatively charged aminoacid at the position corresponding to 281 of SEQ ID NO: 70; c) anegatively charged amino acid at the position corresponding to 360 ofSEQ ID NO: 70; d) a negatively charged amino acid at the positioncorresponding to 281 of SEQ ID NO: 70 and a negatively charged aminoacid at the position corresponding to 360 of SEQ ID NO: 70; e) acysteine at the position corresponding to 275 of SEQ ID NO: 70, atryptophan at the position corresponding to 287 of SEQ ID NO: 70, and anegatively charged amino acid at the position corresponding to 281 ofSEQ ID NO: 70; f) a cysteine at the position corresponding to 275 of SEQID NO: 70, a tryptophan at the position corresponding to 287 of SEQ IDNO: 70, and a negatively charged amino acid at the positioncorresponding to 360 of SEQ ID NO: 70; and g) a cysteine at the positioncorresponding to 275 of SEQ ID NO: 70, a tryptophan at the positioncorresponding to 287 of SEQ ID NO: 70, a negatively charged amino acidat the position corresponding to 281 of SEQ ID NO: 70, and a negativelycharged amino acid at the position corresponding to 360 of SEQ ID NO:70. In some embodiments, the negatively charged amino acid residue is amodified or naturally occurring (e.g., D or E) amino acid. In someembodiments, the negatively charged amino acid residue is D. In someembodiments, the negatively charged amino acid residue is E.

In certain aspects, the disclosure relates to a recombinant ALK4:ActRIIBheteromultimer comprising at least one ALK4-Fc fusion protein and atleast one ActRIIB-Fc fusion protein, wherein the ALK4-Fc fusion proteincomprises an amino acid sequence that is at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% to the amino acid sequence of SEQ ID NO: 48, and wherein theActRIIB-Fc fusion protein comprises an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% to the amino acid sequence of SEQ IDNO: 80. In some embodiments, the ALK4-Fc fusion protein Fc domaincomprises a cysteine at the position corresponding to 234 of SEQ ID NO:48, a serine at the position corresponding to 251 of SEQ ID NO: 48, analanine at the position corresponding to 253 of SEQ ID NO: 48, and avaline at position 292 of SEQ ID NO: 48. In some embodiments, theActRIIB-Fc fusion protein Fc domain comprises a cysteine at the positioncorresponding to 250 of SEQ ID NO: 80, a tryptophan at the positioncorresponding to 262 of SEQ ID NO: 80, and a arginine at the positioncorresponding to 331 of SEQ ID NO: 80.

In certain aspects, the disclosure relates to a recombinant ALK4-Fcfusion protein comprises an amino acid sequence that is at least 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% to the amino acid sequence of SEQ ID NO: 48. Insome embodiments, the ALK4-Fc fusion protein Fc domain comprises acysteine at the position corresponding to 234 of SEQ ID NO: 48, a serineat the position corresponding to 251 of SEQ ID NO: 48, an alanine at theposition corresponding to 253 of SEQ ID NO: 48, and a valine at position292 of SEQ ID NO: 48.

In certain aspects, the disclosure relates to a recombinant ActRIIB-Fcfusion protein comprises an amino acid sequence that is at least 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% to the amino acid sequence of SEQ ID NO: 80. Insome embodiments the ActRIIB-Fc fusion protein Fc domain comprises acysteine at the position corresponding to 250 of SEQ ID NO: 80, atryptophan at the position corresponding to 262 of SEQ ID NO: 80, and aarginine at the position corresponding to 331 of SEQ ID NO: 80.

In certain aspects, the disclosure relates to a recombinant ALK4-Fcfusion protein comprises an amino acid sequence that is at least 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% to the amino acid sequence of SEQ ID NO: 47. Insome embodiments, the ALK4-Fc fusion protein Fc domain comprises acysteine at the position corresponding to 258 of SEQ ID NO: 47, a serineat the position corresponding to 275 of SEQ ID NO: 47, an alanine at theposition corresponding to 277 of SEQ ID NO: 47, and a valine at position316 of SEQ ID NO: 47.

In certain aspects, the disclosure relates to a recombinant ActRIIB-Fcfusion protein comprises an amino acid sequence that is at least 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% to the amino acid sequence of SEQ ID NO: 78. Insome embodiments, the ActRIIB-Fc fusion protein Fc domain comprises acysteine at the position corresponding to 275 of SEQ ID NO: 78, atryptophan at the position corresponding to 287 of SEQ ID NO: 78, and aarginine at the position corresponding to 356 of SEQ ID NO: 78.

In certain aspects, the disclosure relates to a recombinant ALK4:ActRIIBheteromultimer comprising at least one ALK4-Fc fusion protein and atleast one ActRIIB-Fc fusion protein, wherein the ALK4-Fc fusion proteincomprises an amino acid sequence that is at least 90%, 95%, 97% or 99%identical to the amino acid sequence of SEQ ID NO: 76, and wherein theActRIIB-Fc fusion protein comprises an amino acid sequence that is atleast 90%, 95%, 97% or 99% identical to the amino acid sequence of SEQID NO: 72. In some embodiments, the ALK4-Fc fusion protein comprises theamino acid sequence of SEQ ID NO: 76, and the ActRIIB-Fc fusion proteincomprises the amino acid sequence of SEQ ID NO: 72. In some embodiments,the ActRIIB-Fc fusion protein comprises the leader sequence of SEQ IDNO: 38. In some embodiments, the ActRIIB-Fc fusion protein comprises anamino acid sequence that is at least 90%, 95%, 97% or 99% identical tothe amino acid sequence of SEQ ID NO: 70. In some embodiments, theActRIIB-Fc fusion protein comprises the amino acid sequence of SEQ IDNO: 70. In some embodiments, the ALK4-Fc fusion protein comprises theleader sequence of SEQ ID NO: 38. In some embodiments, the ALK4-Fcfusion protein comprises an amino acid sequence that is at least 90%,95%, 97% or 99% identical to the amino acid sequence of SEQ ID NO: 74.In some embodiments, the ALK4-Fc fusion protein comprises the amino acidsequence of SEQ ID NO: 74. In some embodiments, the ALK4-Fc fusionprotein comprises the amino acid sequence of SEQ ID NO: 74, and theActRIIB-Fc fusion protein comprises the amino acid sequence of SEQ IDNO: 70.

Optionally, an ALK4 and/or ActRIIB polypeptide comprises one or moremodified amino acid residues selected from: a glycosylated amino acid, aPEGylated amino acid, a farnesylated amino acid, an acetylated aminoacid, a biotinylated amino acid, an amino acid conjugated to a lipidmoiety, and an amino acid conjugated to an organic derivatizing agent.ALK4 and/or ActRIIB polypeptides may comprise at least one N-linkedsugar, and may include two, three or more N-linked sugars. Suchpolypeptides may also comprise O-linked sugars. ALK4 and/or ActRIIBpolypeptides may be produced in a variety of cell lines that glycosylatethe protein in a manner that is suitable for patient use, includingengineered insect or yeast cells, and mammalian cells such as COS cells,CHO cells, HEK cells and NSO cells. In some embodiments an ALK4 and/orActRIIB polypeptide is glycosylated and has a glycosylation patternobtainable from a Chinese hamster ovary cell line. PreferablyALK4:ActRIIB heteromultimer complexes of the disclosure exhibit a serumhalf-life of at least 4, 6, 12, 24, 36, 48, or 72 hours in a mammal(e.g., a mouse or a human). Optionally, ALK4:ActRIIB heteromultimers mayexhibit a serum half-life of at least 6, 8, 10, 12, 14, 20, 25, or 30days in a mammal (e.g., a mouse or a human).

In certain aspects, ALK4:ActRIIB heteromultimers of the disclosure bindto one or more TGF-beta superfamily ligands. Optionally, ALK4:ActRIIBheteromultimers bind to one or more of these ligands with a K_(D) ofless than or equal to 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, or 10⁻¹² M. For example,in some embodiments, ALK4:ActRIIB heteromultimers bind to activin B. Insome embodiments, ALK4:ActRIIB heteromultimers bind to activin A. Insome embodiments, ALK4:ActRIIB heteromultimers bind to activin AB. Insome embodiments, ALK4:ActRIIB heteromultimers bind to activin C. Insome embodiments, ALK4:ActRIIB heteromultimers bind to activin AC. Insome embodiments, ALK4:ActRIIB heteromultimers bind to activin BC. Insome embodiments, ALK4:ActRIIB heteromultimers bind to activin BC. Insome embodiments, ALK4:ActRIIB heteromultimers bind to activin BE. Insome embodiments, ALK4:ActRIIB heteromultimers bind to GDF11. In someembodiments, ALK4:ActRIIB heteromultimers bind to GDF8. In someembodiments, ALK4:ActRIIB heteromultimers bind to BMP6. In someembodiments, ALK4:ActRIIB heteromultimers bind to GDF3. In someembodiments, ALK4:ActRIIB heteromultimers bind to BMP10. In someembodiments, ALK4:ActRIIB heteromultimers do not bind to, or do notsubstantially bind to, BMP9 (e.g., bind with a K_(D) of greater than orequal to 10⁻⁸ or 10⁻⁷). In some embodiments, ALK4:ActRIIBheteromultimers bind to activin B with stronger affinity compared to acorresponding ActRIIB homomultimer. In some embodiments, ALK4:ActRIIBheteromultimers bind to GDF3 with weaker affinity compared to acorresponding ActRIIB homomultimer. In some embodiments, ALK4:ActRIIBheteromultimers bind to BMP10 with weaker affinity compared to acorresponding ActRIIB homomultimer. In some embodiments, ALK4:ActRIIBheteromultimers bind to BMP9 with weaker affinity compared to acorresponding ActRIIB homomultimer.

In general, ALK4:ActRIIB heteromultimers of the disclosure antagonize(inhibit) one or more activities of at least one TGF-beta superfamilyligand, and such alterations in activity may be measured using variousassays known in the art, including, for example, a cell-based assay suchas those described herein. In certain aspects, ALK4:ActRIIBheteromultimers may be used to inhibit signaling (e.g., Smad 2/3 and/orSmad 1/5/8 signaling) mediated by one or more TGFβ superfamily ligandsin, for example, a cell-based assay. For example, in some embodiments,ALK4:ActRIIB heteromultimers inhibit activin signaling in a cell-basedassay. In some embodiments, ALK4:ActRIIB heteromultimers inhibit activinsignaling in a cell-based assay. In some embodiments, ALK4:ActRIIBheteromultimers inhibit activin A signaling in a cell-based assay. Insome embodiments, ALK4:ActRIIB heteromultimers inhibit activin Bsignaling in a cell-based assay. In some embodiments, ALK4:ActRIIBheteromultimers inhibit activin AB signaling in a cell-based assay. Insome embodiments, ALK4:ActRIIB heteromultimers inhibit activin Csignaling in a cell-based assay. In some embodiments, ALK4:ActRIIBheteromultimers inhibit activin AC signaling in a cell-based assay. Insome embodiments, ALK4:ActRIIB heteromultimers inhibit activin BCsignaling in a cell-based assay. In some embodiments, ALK4:ActRIIBheteromultimers inhibit activin E signaling in a cell-based assay. Insome embodiments, ALK4:ActRIIB heteromultimers inhibit activin AEsignaling in a cell-based assay. In some embodiments, ALK4:ActRIIBheteromultimers inhibit activin CE signaling in a cell-based assay. Insome embodiments, ALK4:ActRIIB heteromultimers inhibit GDF11 signalingin a cell-based assay. In some embodiments, ALK4:ActRIIB heteromultimersinhibit GDF8 signaling in a cell-based assay. In some embodiments,ALK4:ActRIIB heteromultimers inhibit BMP6 signaling in a cell-basedassay. In some embodiments, ALK4:ActRIIB heteromultimers inhibit GDF3signaling in a cell-based assay. In some embodiments, ALK4:ActRIIBheteromultimers inhibit BMP10 signaling in a cell-based assay. In someembodiments, ALK4:ActRIIB heteromultimers does not inhibit, or does notsubstantially inhibit, BMP9 signaling in a cell-based assay. In someembodiments, ALK4:ActRIIB heteromultimers are stronger inhibitors ofactivin B signaling in a cell-based assay. In some embodiments,ALK4:ActRIIB heteromultimers are weaker inhibitors of GDF3 signaling ina cell-based assay. In some embodiments, ALK4:ActRIIB heteromultimersare weaker inhibitors of BMP10 signaling in a cell-based assay. In someembodiments, ALK4:ActRIIB heteromultimers are weaker inhibitors of BMP9signaling in a cell-based assay.

Any of the ALK4:ActRIIB heteromultimers described herein may beformulated as a pharmaceutical preparation (compositions). In someembodiments, pharmaceutical preparations comprise a pharmaceuticallyacceptable carrier. In some embodiments, a pharmaceutical preparationwill be pyrogen-free (meaning pyrogen free to the extent required byregulations governing the quality of products for therapeutic use). Apharmaceutical preparation may also include one or more additionalcompounds such as a compound that is used to treat a disorder/conditiondescribed herein. In general, ALK4:ActRIIB heteromultimer pharmaceuticalpreparations are substantially free of ALK4 and/or ActRIIBhomomultimers. For example, in some embodiments, ALK4:ActRIIBheteromultimer pharmaceutical preparations comprise less than about 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% ALK4homomultimers. In some embodiments, ALK4:ActRIIB heteromultimerpharmaceutical preparations comprise less than about 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, or less than about 1% ActRIIB homomultimers. In someembodiments, ALK4:ActRIIB heteromultimer pharmaceutical preparationscomprise less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or lessthan about 1% ALK4 homomultimers and less than about 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, or less than about 1% ActRIIB homomultimers.

In certain aspects, the disclosure provides nucleic acids encoding anActRIIB polypeptide as described herein. For example, an ActRIIB nucleicacid may comprise of a nucleic acid that is at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to the sequence of 73-396 of SEQ ID NO: 7 or one thathybridizes under stringent conditions to the complement of nucleotides73-396 of SEQ ID NO: 7. Such an nucleic acid may be one that comprisesthe sequence of SEQ ID NOs: 8. In some embodiments, an ActRIIB nucleicacids comprises a nucleotide sequence that is at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 71. In some embodiments, an ActRIIBnucleic acids comprises a nucleotide sequence that is at least 70%, 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to SEQ ID NO: 73. In some embodiments, anActRIIB nucleic acids comprises a nucleotide sequence that is at least70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 79. In someembodiments, an ActRIIB nucleic acids comprises a nucleotide sequencethat is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 81.

In certain aspects, the disclosure provides nucleic acids encoding anActRIIB polypeptide as described herein. For example, an ALK4 nucleicacid may comprise, consists essentially of, or consists of a nucleicacid that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence of 70-378 of SEQ ID NO: 11 or one that hybridizes understringent conditions to the complement of nucleotides 70-378 of SEQ IDNO: 11. In some embodiments, an ALK4 nucleic acids comprises anucleotide sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NO: 75. In some embodiments, an ALK4 nucleic acids comprises anucleotide sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NO: 77. In some embodiments, an ALK4 nucleic acids comprises anucleotide sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NO: 82. In some embodiments, an ALK4 nucleic acids comprises anucleotide sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identicalto SEQ ID NO: 83.

In certain aspects, the present disclosure provides nucleic acidssequence comprising a coding sequence for and ALK4 polypeptide and acoding sequence for the ActRIIB polypeptide. For example, in someembodiments, nucleic acids of the disclosure a) comprises a nucleotidesequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to anyone of SEQ ID NOs: 71, 73, 79, and 77, and b) comprises, consistsessentially of, or consists of a nucleotide sequence that is at least70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID Nos: 75, 77,82, and 83.

Preferably, ALK4 and/or ActRIIB nucleic acids are isolated and/orrecombinant nucleic acids. Nucleic acids disclosed herein may beoperably linked to a promoter for expression. The present disclosurefurther provides vectors comprising such ALK4 and/or ActRIIBpolynucleotides as well as cells (e.g., CHO cells), preferably cellsisolated from a human or other vertebrate species, comprising such ALK4and/or ActRIIB polynucleotides as well as vectors comprising such ALK4and/or ActRIIB polynucleotides.

In certain aspects, an ALK4 polypeptides and/or ActRIIB polypeptides maybe expressed in a mammalian cell line, optionally a cell line thatmediates suitably natural glycosylation of the ActRIIB or ALK4 proteinso as to diminish the likelihood of an unfavorable immune response in apatient (including the possibility of veterinary patients). Human andCHO cell lines have been used successfully, and it is expected thatother common mammalian expression vectors will be useful. Thus thedisclosure provides cultured cells comprising any of the nucleic acidsdisclosed herein. Such cells may be mammalian cells, including CHOcells, NSO cells, HEK cells and COS cells. Other cells may be chosendepending on the species of the intended patient. Other cells aredisclosed herein. Cultured cells are understood to mean cells maintainedin laboratory or other man-made conditions (e.g., frozen, or in media)and not part of a living organism.

In certain aspects, the disclosure provides methods for making any ofthe ALK4 and ActRIIB polypeptides described herein as well asALK4:ActRIIB heteromultimer complexes comprising such polypeptides. Sucha method may include expressing any of the nucleic acids disclosedherein in a suitable cell (e.g., CHO cell or a COS cell). For example,in some embodiments a method of making a heteromultimer comprising anALK4 polypeptide and an ActRIIB polypeptide comprises: culturing a cellunder conditions suitable for expression of an ALK4 polypeptide and anActRIIB polypeptide, wherein the cell comprises an ALK4 polynucleotideand an ActRIIB polynucleotide; optionally recovering the heteromultimerso expressed. Alternatively, a method of making a heteromultimercomprising an ALK4 polypeptide and an ActRIIB polypeptide may comprise:a) culturing a first cell under conditions suitable for expression of anALK4 polypeptide, wherein the first cell comprises an ALK4polynucleotide; b) recovering the ALK4 polypeptide so expressed; c)culturing a second cell under conditions suitable for expression of anActRIIB polypeptide, wherein the second cell comprises an ActRIIBpolynucleotide; d) recovering the ActRIIB polypeptide so expressed; e)combining the recovered ALK4 polypeptide and the recovered ActRIIBpolypeptide under conditions suitable for ALK4:ActRIIB heteromultimerformation; optionally recovering the ALK4:ActRIIB heteromultimer. Incertain embodiments, ALK4 and/or ActRIIB polypeptides are expressedusing a TPA leader sequence (e.g., SEQ ID NO: 38). In certainembodiments, ALK4 and/or ActRIIB polypeptides are expressed in a CHOcell. ALK4 and ActRIIB polypeptides described herein, as well as proteincomplexes of the same, may be recovered as crude, partially purified, orhighly purified fractions using any of the well-known techniques forobtaining protein from cell cultures. In general, such methods result inALK4:ActRIIB heteromultimers that substantially free of ALK4 and/orActRIIB homomultimers. For example, in some embodiments, methods forproducing ALK4:ActRIIB heteromultimers result in less than about 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than about 1% ALK4homomultimers. In some embodiments, methods for producing ALK4:ActRIIBheteromultimers result in less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%,3%, 2%, or less than about 1% ActRIIB homomultimers. In someembodiments, methods for producing ALK4:ActRIIB heteromultimers resultin less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less thanabout 1% ALK4 homomultimers and less than about 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, or less than about 1% ActRIIB homomultimers.

The disclosure further provides methods and ALK4:ActRIIB heteromultimersfor use in the treatment or prevention of variousALK4:ActRIIB-associated diseases and conditions associated with, forexample, muscle, bone, fat, red blood cells, and other tissues. Suchdisease and disorders include, but are not limited to, disordersassociated with muscle loss or insufficient muscle growth (e.g., muscleatrophy; muscular dystrophy, including Duchenne muscular dystrophy,Becker muscular dystrophy, and facioscapulohumeral muscular dystrophy;amyotrophic lateral sclerosis; sporadic inclusion body myositis,hereditary inclusion body myositis, and cachexia) and disordersassociated with undesirable weight gain (e.g., obesity, type 2 diabetesor non-insulin dependent diabetes mellitus (NIDDM), cardiovasculardisease, hypertension, osteoarthritis, stroke, respiratory problems, andgall bladder disease). In some embodiments, ALK4:ActRIIB heteromultimersmay be used to decrease body fat content or reduce the rate of increasein body fat content in a subject in need thereof. In some embodiments,ALK4:ActRIIB heteromultimers may be used to reduce cholesterol and/ortriglyceride levels in a patient. In some embodiments, ALK4:ActRIIBheteromultimers may be used to treat or prevent fibrosis or afibrosis-associated disorder or condition (e.g., renal failure, chronicrenal disease, cystic fibrosis, and myelofibrosis).

The disclosure further provides methods and ALK4:ActRIIB heteromultimersfor use in the treatment or prevention of variousALK4:ActRIIB-associated diseases and conditions associated with, forexample, the kidney. Such diseases or conditions include, for example,chronic kidney disease or failure, acute kidney disease or failure,patients that have stage 1 kidney disease, patients that have stage 2kidney disease, patients that have stage 3 kidney disease, patients thathave stage 4 kidney disease, patients that have stage 5 kidney disease,non-diabetic kidney diseases, glomerulonephritis, interstitialnephritis, diabetic kidney diseases, diabetic nephropathy,glomerulosclerosis, rapid progressive glomerulonephritis, renalfibrosis, Alport syndrome, IDDM nephritis, mesangial proliferativeglomerulonephritis, membranoproliferative glomerulonephritis, crescenticglomerulonephritis, renal interstitial fibrosis, focal segmentalglomerulosclerosis, membranous nephropathy, minimal change disease,pauci-immune rapid progressive glomerulonephritis, IgA nephropathy,polycystic kidney disease, Dent's disease, nephrocytinosis, Heymannnephritis, autosomal dominant (adult) polycystic kidney disease,autosomal recessive (childhood) polycystic kidney disease, acute kidneyinjury, nephrotic syndrome, renal ischemia, podocyte diseases ordisorders, proteinuria, glomerular diseases, membranousglomerulonephritis, focal segmental glomerulonephritis, pre-eclampsia,eclampsia, kidney lesions, collagen vascular diseases, benignorthostatic (postural) proteinuria, IgM nephropathy, membranousnephropathy, sarcoidosis, diabetes mellitus, kidney damage due to drugs,Fabry's disease, aminoaciduria, Fanconi syndrome, hypertensivenephrosclerosis, interstitial nephritis, Sickle cell disease,hemoglobinuria, myoglobinuria, Wegener's Granulomatosis, GlycogenStorage Disease Type 1, chronic kidney disease, chronic renal failure,low Glomerular Filtration Rate (GFR), nephroangiosclerosis, lupusnephritis, ANCA-positive pauci-immune crescentic glomerulonephritis,chronic allograft nephropathy, nephrotoxicity, renal toxicity, kidneynecrosis, kidney damage, glomerular and tubular injury, kidneydysfunction, nephritic syndrome, acute renal failure, chronic renalfailure, proximal tubal dysfunction, acute kidney transplant rejection,chronic kidney transplant rejection, non-IgA mesangioproliferativeglomerulonephritis, postinfectious glomerulonephritis, vasculitides withrenal involvement of any kind, any hereditary renal disease, anyinterstitial nephritis, renal transplant failure, kidney cancer, kidneydisease associated with other conditions (e.g., hypertension, diabetes,and autoimmune disease), Dent's disease, nephrocytinosis, Heymannnephritis, a primary kidney disease, a collapsing glomerulopathy, adense deposit disease, a cryoglobulinemia-associated glomerulonephritis,an Henoch-Schonlein disease, a postinfectious glomerulonephritis, abacterial endocarditis, a microscopic polyangitis, a Churg-Strausssyndrome, an anti-GBM-antibody mediated glomerulonephritis, amyloidosis,a monoclonal immunoglobulin deposition disease, a fibrillaryglomerulonephritis, an immunotactoid glomerulopathy, ischemic tubularinjury, a medication-induced tubulo-interstitial nephritis, a toxictubulo-interstitial nephritis, an infectious tubulo-interstitialnephritis, a bacterial pyelonephritis, a viral infectioustubulo-interstitial nephritis which results from a polyomavirusinfection or an HIV infection, a metabolic-induced tubulo-interstitialdisease, a mixed connective disease, a cast nephropathy, a crystalnephropathy which may results from urate or oxalate or drug-inducedcrystal deposition, an acute cellular tubulo-interstitial allograftrejection, a tumoral infiltrative disease which results from a lymphomaor a post-transplant lymphoproliferative disease, an obstructive diseaseof the kidney, vascular disease, a thrombotic microangiopathy, anephroangiosclerosis, an atheroembolic disease, a mixed connectivetissue disease, a polyarteritis nodosa, a calcineurin-inhibitorinduced-vascular disease, an acute cellular vascular allograftrejection, an acute humoral allograft rejection, early renal functiondecline (ERFD), end stage renal disease (ESRD), renal vein thrombosis,acute tubular necrosis, renal occlusion, acute interstitial nephritis,established chronic kidney disease, renal artery stenosis, ischemicnephropathy, uremia, drug and toxin-induced chronic tubulointerstitialnephritis, reflux nephropathy, kidney stones, Goodpasture's syndrome,normocytic normochromic anemia, renal anemia, diabetic chronic kidneydisease, IgG4-related disease, von Hippel-Lindau syndrome, tuberoussclerosis, nephronophthisis, medullary cystic kidney disease, renal cellcarcinoma, adenocarcinoma, nephroblastoma, lymphoma, leukemia,hyposialylation disorder, chronic cyclosporine nephropathy, renalreperfusion injury, renal dysplasia, azotemia, bilateral arterialocclusion, acute uric acid nephropathy, hypovolemia, acute bilateralobstructive uropathy, hypercalcemic nephropathy, hemolytic uremicsyndrome, acute urinary retention, malignant nephrosclerosis, postpartumglomerulosclerosis, scleroderma, non-Goodpasture's anti-GBM disease,microscopic polyarteritis nodosa, allergic granulomatosis, acuteradiation nephritis, post-streptococcal glomerulonephritis,Waldenstrom's macroglobulinemia, analgesic nephropathy, arteriovenousfistula, arteriovenous graft, dialysis, ectopic kidney, medullary spongekidney, renal osteodystrophy, solitary kidney, hydronephrosis,microalbuminuria, uremia, haematuria, hyperlipidemia, hypoalbuminaemia,lipiduria, acidosis, and hyperkalemia. In some embodiments, thedisclosure further provides methods and ALK4:ActRIIB antagonists for usein delaying or preventing progression from: stage 1 to stage 2 kidneydisease, stage 2 to stage 3 kidney disease, stage 3 to stage 4 kidneydisease, or stage 4 to stage 5 kidney disease.

In some embodiments, the disclosure further provides methods andALK4:ActRIIB heteromultimers for use in preventing or reducing kidneyinflammation. In some embodiments, the disclosure further providesmethods and ALK4:ActRIIB heteromultimers for use in preventing orreducing kidney damage. In some embodiments, the disclosure furtherprovides methods and ALK4:ActRIIB heteromultimers for use in preventingor reducing kidney fibrosis.

In certain aspects, the disclosure relates to methods of treatingpulmonary arterial hypertension comprising administering to a patient inneed thereof an effective amount of an ALK4:ActRIIB heteromultimer suchas those described herein (e.g., an ALK4:ActRIIB heterodimer). In someembodiments, administration of the ALK4:ActRIIB heteromultimer decreasesventricle hypertrophy in the patient. In some embodiments,administration of the ALK4:ActRIIB heteromultimer decreases ventriclehypertrophy in the patient by at least 10% (e.g., 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80%). Insome embodiments, administration of the ALK4:ActRIIB heteromultimerdecreases smooth muscle hypertrophy in the patient. In some embodiments,administration of the ALK4:ActRIIB heteromultimer decreases smoothmuscle hypertrophy in the patient by at least 10% (e.g., 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80%).In some embodiments, administration of the ALK4:ActRIIB heteromultimerdecreases pulmonary arteriole muscularity in the patient. In someembodiments, administration of the ALK4:ActRIIB heteromultimer decreasespulmonary arteriole muscularity in the patient by at least 10% (e.g.,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, orat least 80%). In some embodiments, administration of the ALK4:ActRIIBheteromultimer decreases pulmonary vascular resistance in the patient.In some embodiments, administration of the ALK4:ActRIIB heteromultimerdecreases pulmonary vascular resistance in the patient by at least 10%(e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, or at least 80%). In some embodiments, administration of theALK4:ActRIIB heteromultimer decreases pulmonary vascular resistance inthe patient by at least 25-30%. In some embodiments, the patient haspulmonary arterial hypertension and has Functional Class II or Class IIIpulmonary hypertension in accordance with the World HealthOrganization's functional classification system for pulmonaryhypertension. In some embodiments, the patient has pulmonary arterialhypertension that is classified as one or more subtypes selected fromthe group consisting of: idiopathic or heritable pulmonary arterialhypertension, drug- and/or toxin-induced pulmonary hypertension,pulmonary hypertension associated with connective tissue disease, andpulmonary hypertension associated with congenital systemic-to-pulmonaryshunts at least 1 year following shunt repair. In some embodiments, thepatient has been treated with one or more vasodilators. In someembodiments, the patient has been treated with one or more agentsselected from the group consisting of: phosphodiesterase type 5inhibitors, soluble guanylate cyclase stimulators, prostacyclin receptoragonist, and endothelin receptor antagonists. In some embodiments, theone or more agents is selected from the group consisting of: bosentan,sildenafil, beraprost, macitentan, selexipag, epoprostenol,treprostinil, iloprost, ambrisentan, and tadalafil. In some embodiments,the method further comprises administration of one or more vasodilators.In some embodiments, the method further comprises administration of oneor more agents selected from the group consisting of: phosphodiesterasetype 5 inhibitors, soluble guanylate cyclase stimulators, prostacyclinreceptor agonist, and endothelin receptor antagonists. In someembodiments, the one or more agents is selected from the groupconsisting of: bosentan, sildenafil, beraprost, macitentan, selexipag,epoprostenol, treprostinil, iloprost, ambrisentan, and tadalafil. Insome embodiments, the patient has a 6-minute walk distance from 150 to400 meters. In some embodiments, the method increases the patient's6-minute walk distance by at least 10 meters (e.g., at least 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, or more than400 meters). In some embodiments, the patient has a hemoglobin levelfrom >8 and <15 g/dl. In some embodiments, the method delays clinicalworsening of pulmonary arterial hypertension. In some embodiments, themethod delays clinical worsening of pulmonary hypertension in accordancewith the World Health Organization's functional classification systemfor pulmonary hypertension. In some embodiments, the method reduces therisk of hospitalization for one or more complications associated withpulmonary arterial hypertension.

In some embodiments, the present disclosure relates to methods oftreating pulmonary hypertension comprising administering to a patient inneed thereof an effective amount of an ALK4:ActRIIB heteromultimer. Incertain aspects, the disclosure relates to methods of preventingpulmonary hypertension comprising administering to a patient in needthereof an effective amount of an ALK4:ActRIIB heteromultimer. Incertain aspects, the disclosure relates to methods of reducing theprogression rate of pulmonary hypertension comprising administering to apatient in need thereof an effective amount of an ALK4:ActRIIBheteromultimer. In some embodiments, the disclosure provides for amethod of treating an interstitial lung disease, comprisingadministering to a patient in need thereof an effective amount of anALK4:ActRIIB heteromultimer. In some embodiments, the disclosureprovides for a method of treating, preventing, or reducing theprogression rate and/or severity of one or more complications of aninterstitial lung disease, comprising administering to a patient in needthereof an effective amount of an ALK4:ActRIIB heteromultimer. In someembodiments, the interstitial lung disease is idiopathic pulmonaryfibrosis. In certain aspects, the disclosure relates to methods ofreducing the severity of pulmonary hypertension comprising administeringto a patient in need thereof an effective amount of an ALK4:ActRIIBheteromultimer. In certain aspects, the disclosure relates to methods oftreating one or more complications (e.g., smooth muscle and/orendothelial cell proliferation in the pulmonary artery, angiogenesis inthe pulmonary artery, dyspnea, chest pain, pulmonary vascularremodeling, right ventricular hypertrophy, and pulmonary fibrosis) ofpulmonary hypertension comprising administering to a patient in needthereof an effective amount of an ALK4:ActRIIB heteromultimer. Incertain aspects, the disclosure relates to methods of preventing one ormore complication of pulmonary hypertension (e.g., smooth muscle and/orendothelial cell proliferation in the pulmonary artery, angiogenesis inthe pulmonary artery, dyspnea, chest pain, pulmonary vascularremodeling, right ventricular hypertrophy, and pulmonary fibrosis)comprising administering to a patient in need thereof an effectiveamount an ALK4:ActRIIB heteromultimer. In certain aspects, thedisclosure relates to methods of reducing the progression rate of one ormore complication of pulmonary hypertension (e.g., smooth muscle and/orendothelial cell proliferation in the pulmonary artery, angiogenesis inthe pulmonary artery, dyspnea, chest pain, pulmonary vascularremodeling, right ventricular hypertrophy, and pulmonary fibrosis)comprising administering to a patient in need thereof an effectiveamount an ALK4:ActRIIB heteromultimer. In certain aspects, thedisclosure relates to methods of reducing the severity of one or morecomplication of pulmonary hypertension (e.g., smooth muscle and/orendothelial cell proliferation in the pulmonary artery, angiogenesis inthe pulmonary artery, dyspnea, chest pain, pulmonary vascularremodeling, right ventricular hypertrophy, and pulmonary fibrosis)comprising administering to a patient in need thereof an effectiveamount of an ALK4:ActRIIB heteromultimer. In certain preferredembodiments, methods described herein relate to a patient havingpulmonary arterial hypertension. In some embodiments, methods describedherein relate to a patient having a resting pulmonary arterial pressure(PAP) of at least 25 mm Hg (e.g., at least 25, 30, 35, 40, 45, or 50 mmHg). In some embodiments, the methods described herein reduce PAP in apatient having pulmonary hypertension. For example, the method mayreduce PAP by at least 3 mmHg (e.g., at least 3, 5, 7, 10, 12, 15, 20,or 25 mm Hg) in a patient having pulmonary hypertension. In someembodiments, the methods described herein reduce pulmonary vascularresistance in a patient having pulmonary hypertension. In someembodiments, the methods described herein increase pulmonary capillarywedge pressure in a patient having pulmonary hypertension. In someembodiments, the methods described herein increase left ventricularend-diastolic pressure in a patient having pulmonary hypertension. Insome embodiments, the methods described herein increase (improves)exercise capacity (ability, tolerance) in a patient having pulmonaryhypertension. For example, the method may increase 6-minute walkdistance in a patient having pulmonary hypertension, optionallyincreasing 6-minute walk distance by at least 10 meters (e.g., at least10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more meters). In addition,the method may reduce the patient's Borg dyspnea index (BDI), whichoptionally may be assessed after a 6-minute walk test. In someembodiments, the method reduces the patient's Borg dyspnea index (BDI)by at least 0.5 index points (e.g., at least 0.5, 1, 1.5, 2, 2.5, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 indexpoints). In some embodiments, the methods described herein relate to apatient having Class I, Class II, Class III, or Class IV pulmonaryhypertension as recognized by the World Health Organization. In someembodiments, the methods described herein relate to delaying clinicalprogression (worsening) of pulmonary hypertension (e.g., progression asmeasured by the World Health Organization standard). In someembodiments, the method prevents or delays pulmonary hypertension Classprogression (e.g., prevents or delays progression from Class I to ClassII, Class II to Class III, or Class III to Class IV pulmonaryhypertension as recognized by the World Health Organization). In someembodiments, the method promotes or increases pulmonary hypertensionClass regression (e.g., promotes or increases regression from Class IVto Class III, Class III to Class II, or Class II to Class I pulmonaryhypertension as recognized by the World Health Organization). In someembodiments, the patient is further administered one or more supportivetherapies or active agents for treating pulmonary hypertension inaddition to the one or more GDF/BMP antagonist. For example, the patientalso may be administered one or more supportive therapies or activeagents selected from the group consisting of: prostacyclin andderivatives thereof (e.g., epoprostenol, treprostinil, and iloprost);prostacyclin receptor agonists (e.g., selexipag); endothelin receptorantagonists (e.g., thelin, ambrisentan, macitentan, and bosentan);calcium channel blockers (e.g., amlodipine, diltiazem, and nifedipine;anticoagulants (e.g., warfarin); diuretics; oxygen therapy; atrialseptostomy; pulmonary thromboendarterectomy; phosphodiesterase type 5inhibitors (e.g., sildenafil and tadalafil); activators of solubleguanylate cyclase (e.g., cinaciguat and riociguat); ASK-1 inhibitors(e.g., CIIA; SCH79797; GS-4997; MSC2032964A;3H-naphtho[1,2,3-de]quiniline-2,7-diones, NQDI-1;2-thioxo-thiazolidines,5-bromo-3-(4-oxo-2-thioxo-thiazolidine-5-ylidene)-1,3-dihydro-indol-2-one);NF-κB antagonists (e.g., dh404, CDDO-epoxide; 2.2-difluoropropionamide;C28 imidazole (CDDO-Im); 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid(CDDO); 3-Acetyloleanolic Acid; 3-Triflouroacetyloleanolic Acid;28-Methyl-3-acetyloleanane; 28-Methyl-3-trifluoroacetyloleanane;28-Methyloxyoleanolic Acid; SZC014; SCZ015; SZC017; PEGylatedderivatives of oleanolic acid; 3-O-(beta-D-glucopyranosyl) oleanolicacid; 3-O-[beta-D-glucopyranosyl-(1→3)-beta-D-glucopyranosyl] oleanolicacid; 3-O-[beta-D-glucopyranosyl-(1→2)-beta-D-glucopyranosyl] oleanolicacid; 3-O-[beta-D-glucopyranosyl-(1→3)-beta-D-glucopyranosyl] oleanolicacid 28-O-beta-D-glucopyranosyl ester;3-O-[beta-D-glucopyranosyl-(1→2)-beta-D-glucopyranosyl] oleanolic acid28-O-beta-D-glucopyranosyl ester;3-O-[a-L-rhamnopyranosyl-(1→3)-beta-D-glucuronopyranosyl] oleanolicacid; 3-O-[alpha-L-rhamnopyranosyl-(1→3)-beta-D-glucuronopyranosyl]oleanolic acid 28-O-beta-D-glucopyranosyl ester;28-O-β-D-glucopyranosyl-oleanolic acid; 3-O-β-D-glucopyranosyl(1→3)-β-D-glucopyranosiduronic acid (CS1); oleanolic acid3-O-β-D-glucopyranosyl (1→3)-β-D-glucopyranosiduronic acid (CS2); methyl3,11-dioxoolean-12-en-28-olate (DIOXOL); ZCVI₄-2; Benzyl3-dehydr-oxy-1,2,5-oxadiazolo[3′,4′:2,3]oleanolate) lung and/or hearttransplantation.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof necessary fee.

FIG. 1 shows an alignment of extracellular domains of human ActRIIA (SEQID NO: 49) and human ActRIIB (SEQ ID NO: 2) with the residues that arededuced herein, based on composite analysis of multiple ActRIIB andActRIIA crystal structures, to directly contact ligand indicated withboxes.

FIG. 2 shows a multiple sequence alignment of various vertebrate ActRIIBprecursor proteins without their intracellular domains (SEQ ID NOs:50-55, respectively) human ActRIIA precursor protein without itsintracellular domain (SEQ ID NO: 56), and a consensus ActRII precursorprotein (SEQ ID NO: 57).

FIG. 3 shows multiple sequence alignment of Fc domains from human IgGisotypes using Clustal 2.1. Hinge regions are indicated by dottedunderline. Double underline indicates examples of positions engineeredin IgG1 Fc (SEQ ID NO: 31) to promote asymmetric chain pairing and thecorresponding positions with respect to other isotypes IgG2 (SEQ ID NO:32), IgG3 (SEQ ID NO: 33) and IgG4 (SEQ ID NO: 35).

FIG. 4 shows comparative ligand binding data for an ALK4-Fc:ActRIIB-Fcheterodimeric protein complex compared to ActRIIB-Fc homodimer andALK4-Fc homodimer. For each protein complex, ligands are ranked byk_(off), a kinetic constant that correlates well with ligand signalinginhibition, and listed in descending order of binding affinity (ligandsbound most tightly are listed at the top). At left, yellow, red, green,and blue lines indicate magnitude of the off-rate constant. Solid blacklines indicate ligands whose binding to heterodimer is enhanced orunchanged compared with homodimer, whereas dashed red lines indicatesubstantially reduced binding compared with homodimer. As shown, theALK4-Fc:ActRIIB-Fc heterodimer displays enhanced binding to activin Bcompared with either homodimer, retains strong binding to activin A,GDF8, and GDF11 as observed with ActRIIB-Fc homodimer, and exhibitssubstantially reduced binding to BMP9, BMP10, and GDF3. Like ActRIIB-Fchomodimer, the heterodimer retains intermediate-level binding to BMP6.

FIG. 5 shows a multiple sequence alignment of ALK4 extracellular domainsderived from various vertebrate species (SEQ ID NOs: 59-65).

FIGS. 6A-6D show schematic examples of heteromeric protein complexescomprising an ALK4 polypeptide and an ActRIIB polypeptide.

In the illustrated embodiments, the ALK4 polypeptide (from left toright) is part of a fusion polypeptide that comprises a first member ofan interaction pair (“C₁”), and the ActRIIB polypeptide is part of afusion polypeptide that comprises a second member of an interaction pair(“C₂”). Suitable interaction pairs included, for example, heavy chainand/or light chain immunoglobulin interaction pairs, truncations, andvariants thereof such as those described herein [e.g., Spiess et al(2015) Molecular Immunology 67(2A): 95-106]. In each fusion polypeptide,a linker may be positioned between the ALK4 or ActRIIB polypeptide andthe corresponding member of the interaction pair. The first and secondmembers of the interaction pair may be unguided, meaning that themembers of the pair may associate with each other or self-associatewithout substantial preference, and they may have the same or differentamino acid sequences. See FIG. 6A. Alternatively, the interaction pairmay be a guided (asymmetric) pair, meaning that the members of the pairassociate preferentially with each other rather than self-associate. SeeFIG. 6B. Complexes of higher order can be envisioned. See FIGS. 6C and6D.

FIG. 7 shows comparative ALK4-Fc:ActRIIB-Fcheterodimer/ActRIIB-Fc:ActRIIB-Fc homodimer IC₅₀ data as determined byan A-204 Reporter Gene Assay as described herein. ALK4-Fc:ActRIIB-Fcheterodimer inhibits activin A, activin B, GDF8, and GDF11 signalingpathways similarly to the ActRIIB-Fc:ActRIIB-Fc heterodimer. However,ALK4-Fc:ActRIIB-Fc heterodimer inhibition of BMP9 and BMP10 signalingpathways is significantly reduced compared to the ActRIIB-Fc:ActRIIB-Fcheterodimer. These data demonstrate that ALK4:ActRIIB heterodimers aremore selective antagonists of activin A, activin B, GDF8, and GDF11compared to corresponding ActRIIB:ActRIIB homodimers.

FIGS. 8A-8C shows gene expression profiles of fibrotic genes (Col1a1,Fibronectin, PAI-1, CTGF, and a-SMA), inflammatory genes (TNF-alpha, andMCP1), cytokine genes (TGF-beta 1, GF-beta 2, TGF-beta 3, and activinA), kidney injury gene (NGAL), Hypoxia-inducible factor 1-alpha (HIF1a),and activin A receptor (Acvr2A) from mouse kidneys subjected tounilateral ureteral obstruction (UUO). Samples from the contralateral,non-surgery kidney were used as a control (Ctrl). Gene expressionprofiles were obtained at 17 days post-surgery. Mice were administeredeither PBS or an ALK4-Fc:ActRIIB-Fc homodimer at days 3, 7, 10, and 14post-surgery. ($) denotes a statistical difference between UUO kidneysat 17 days in mice administered only PBS compared UUO kidneys at 17 daysin mice administered the ALK7-Fc:ActRIIB-Fc homodimer. (@) denotes thatno transcript was detected.

FIGS. 9A and 9B shows proteinuria levels in Col4a3 and Col4a5 Alportmice treated with vehicle (Col4a3-Veh and Col4a5-Veh) or anALK4-Fc:ActRIIB-Fc fusion protein (Col4a3-IIB/ALK4 and Col4a5-IIB/ALK4).FIG. 9A shows that ALK4-Fc:ActRIIB-Fc treatment significantly reducedproteinuria levels compared to vehicle in the Col4a3 Alport mice. FIG.9B shows that ALK4-Fc:ActRIIB-Fc treatment significantly reducedproteinuria levels compared to vehicle in the Col4a5 Alport mice.

FIGS. 10A-10C show histological analysis of kidney tissue from Col4a3−/−Alport mice treated with vehicle (Col4a3-Veh) or an ALK4-Fc:ActRIIB-Fcheterodimer (Col4a3-IIB/ALK4). FIG. 10A represents the percentage oftissue fibrosis as revealed by collagen-I staining. Collagen-I stainingindicates that ALK4-Fc:ActRIIB-Fc treatment significantly reduced kidneyfibrosis in this Alport mouse model. FIG. 10B represents the percentageof tissue fibrosis as revealed by trichrome staining. Trichrome stainingindicates that ALK4-Fc:ActRIIB-Fc treatment significantly reduced kidneyfibrosis in this Alport mouse model.

FIG. 10C represents the percentage of sclerotic glomeruli as revealed byhistological analysis. The data indicate that ALK4-Fc:ActRIIB-Fctreatment significantly reduced sclerotic glomeruli in this Alport mousemodel.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

In part, the present disclosure relates to heteromultimers comprising aTGFβ superfamily type I receptor polypeptide and a TGFβ superfamily typeII receptor polypeptide, uses thereof, and methods of making suchheteromultimers. See, e.g., FIG. 6. In certain preferred embodiments,heteromultimers comprise an extracellular domain of a TGFβ superfamilytype I receptor polypeptide and an extracellular domain of a TGFβsuperfamily type II receptor polypeptide. In particular, the disclosureprovides heteromultimers that comprise an ALK4 polypeptide and anActRIIB polypeptide. Preferably such ALK4 polypeptides comprise aligand-binding domain of an ALK4 receptor and such ActRIIB polypeptidescomprise a ligand-binding domain of an ActRIIB receptor. In certainpreferred embodiments, ALK4:ActRIIB heteromultimers of the disclosurehave an altered TGFβ superfamily ligand binding profile/specificitycompared to a corresponding sample of a homomultimer (e.g., anALK4:ActRIIB heterodimer compared to an ActRIIB:ActRIIB homodimer or anALK4:ALK4 homodimer).

The TGF-β superfamily is comprised of over 30 secreted factors includingTGF-betas, activins, nodals, bone morphogenetic proteins (BMPs), growthand differentiation factors (GDFs), and anti-Mullerian hormone (AMH)[Weiss et al. (2013) Developmental Biology, 2(1): 47-63]. Members of thesuperfamily, which are found in both vertebrates and invertebrates, areubiquitously expressed in diverse tissues and function during theearliest stages of development throughout the lifetime of an animal.Indeed, TGF-β superfamily proteins are key mediators of stem cellself-renewal, gastrulation, differentiation, organ morphogenesis, andadult tissue homeostasis. Consistent with this ubiquitous activity,aberrant TGF-beta superfamily signaling is associated with a wide rangeof human pathologies including, for example, autoimmune disease,cardiovascular disease, fibrotic disease, and cancer.

Ligands of the TGF-beta superfamily share the same dimeric structure inwhich the central 3½ turn helix of one monomer packs against the concavesurface formed by the beta-strands of the other monomer. The majority ofTGF-beta family members are further stabilized by an intermoleculardisulfide bond. This disulfide bonds traverses through a ring formed bytwo other disulfide bonds generating what has been termed a ‘cysteineknot’ motif [Lin et al. (2006) Reproduction 132: 179-190; and Hinck etal. (2012) FEBS Letters 586: 1860-1870].

TGF-beta superfamily signaling is mediated by heteromeric complexes oftype I and type II serine/threonine kinase receptors, whichphosphorylate and activate downstream SMAD proteins (e.g., SMAD proteins1, 2, 3, 5, and 8) upon ligand stimulation [Massagué (2000) Nat. Rev.Mol. Cell Biol. 1:169-178]. These type I and type II receptors aretransmembrane proteins, composed of a ligand-binding extracellulardomain with cysteine-rich region, a transmembrane domain, and acytoplasmic domain with predicted serine/threonine kinase specificity.In general, type I receptors mediate intracellular signaling while thetype II receptors are required for binding TGF-beta superfamily ligands.Type I and II receptors form a stable complex after ligand binding,resulting in phosphorylation of type I receptors by type II receptors.

The TGF-beta family can be divided into two phylogenetic branches basedon the type I receptors they bind and the Smad proteins they activate.One is the more recently evolved branch, which includes, e.g., theTGF-betas, activins, GDF8, GDF9, GDF11, BMP3 and nodal, which signalthrough type I receptors that activate Smads 2 and 3 [Hinck (2012) FEBSLetters 586:1860-1870]. The other branch comprises the more distantlyrelated proteins of the superfamily and includes, e.g., BMP2, BMP4,BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF1, GDF5, GDF6, and GDF7,which signal through Smads 1, 5, and 8.

Activins are members of the TGF-beta superfamily and were initiallydiscovered as regulators of secretion of follicle-stimulating hormone,but subsequently various reproductive and non-reproductive roles havebeen characterized. There are three principal activin forms (A, B, andAB) that are homo/heterodimers of two closely related β subunits(β_(A)β_(A), β_(B)β_(B), and PAN, respectively). The human genome alsoencodes an activin C and an activin E, which are primarily expressed inthe liver, and heterodimeric forms containing β_(C) or β_(E) are alsoknown. In the TGF-beta superfamily, activins are unique andmultifunctional factors that can stimulate hormone production in ovarianand placental cells, support neuronal cell survival, influencecell-cycle progress positively or negatively depending on cell type, andinduce mesodermal differentiation at least in amphibian embryos [DePaoloet al. (1991) Proc Soc Ep Biol Med. 198:500-512; Dyson et al. (1997)Curr Biol. 7:81-84; and Woodruff (1998) Biochem Pharmacol. 55:953-963].In several tissues, activin signaling is antagonized by its relatedheterodimer, inhibin. For example, in the regulation offollicle-stimulating hormone (FSH) secretion from the pituitary, activinpromotes FSH synthesis and secretion, while inhibin reduces FSHsynthesis and secretion. Other proteins that may regulate activinbioactivity and/or bind to activin include follistatin (FS),follistatin-related protein (FSRP, also known as FLRG or FSTL3), andα₂-macroglobulin.

As described herein, agents that bind to “activin A” are agents thatspecifically bind to the β_(A) subunit, whether in the context of anisolated β_(A) subunit or as a dimeric complex (e.g., a PAPA homodimeror a β_(A)β_(B) heterodimer). In the case of a heterodimer complex(e.g., a β_(A)β_(B) heterodimer), agents that bind to “activin A” arespecific for epitopes present within the β_(A) subunit, but do not bindto epitopes present within the non-β_(A) subunit of the complex (e.g.,the β_(B) subunit of the complex). Similarly, agents disclosed hereinthat antagonize (inhibit) “activin A” are agents that inhibit one ormore activities as mediated by a β_(A) subunit, whether in the contextof an isolated β_(A) subunit or as a dimeric complex (e.g., a β_(A)β_(A)homodimer or a β_(A)β_(B) heterodimer). In the case of β_(A)β_(B)heterodimers, agents that inhibit “activin A” are agents thatspecifically inhibit one or more activities of the β_(A) subunit, but donot inhibit the activity of the non-β_(A) subunit of the complex (e.g.,the β_(B) subunit of the complex). This principle applies also to agentsthat bind to and/or inhibit “activin B”, “activin C”, and “activin E”.Agents disclosed herein that antagonize “activin AB” are agents thatinhibit one or more activities as mediated by the β_(A) subunit and oneor more activities as mediated by the β_(B) subunit. The same principlealso applies to agent that bind to and/or inhibit “activin AC”, “activinBC”, “activin AE”, and “activin BE”.

The BMPs and GDFs together form a family of cysteine-knot cytokinessharing the characteristic fold of the TGF-beta superfamily [Rider etal. (2010) Biochem J., 429(1):1-12]. This family includes, for example,BMP2, BMP4, BMP6, BMP7, BMP2a, BMP3, BMP3b (also known as GDF10), BMP4,BMP5, BMP6, BMP7, BMP8, BMP8a, BMP8b, BMP9 (also known as GDF2), BMP10,BMP11 (also known as GDF11), BMP12 (also known as GDF7), BMP13 (alsoknown as GDF6), BMP14 (also known as GDF5), BMP15, GDF1, GDF3 (alsoknown as VGR2), GDF8 (also known as myostatin), GDF9, GDF15, anddecapentaplegic. Besides the ability to induce bone formation, whichgave the BMPs their name, the BMP/GDFs display morphogenetic activitiesin the development of a wide range of tissues. BMP/GDF homo- andhetero-dimers interact with combinations of type I and type II receptordimers to produce multiple possible signaling complexes, leading to theactivation of one of two competing sets of SMAD transcription factors.BMP/GDFs have highly specific and localized functions. These areregulated in a number of ways, including the developmental restrictionof BMP/GDF expression and through the secretion of several specific BMPantagonist proteins that bind with high affinity to the cytokines.Curiously, a number of these antagonists resemble TGF-beta superfamilyligands.

Growth and differentiation factor-8 (GDF8) is also known as myostatin.GDF8 is a negative regulator of skeletal muscle mass and is highlyexpressed in developing and adult skeletal muscle. The GDF8 nullmutation in transgenic mice is characterized by a marked hypertrophy andhyperplasia of skeletal muscle [McPherron et al. Nature (1997)387:83-90]. Similar increases in skeletal muscle mass are evident innaturally occurring mutations of GDF8 in cattle and, strikingly, inhumans [Ashmore et al. (1974) Growth, 38:501-507; Swatland and Kieffer,J. Anim. Sci. (1994) 38:752-757; McPherron and Lee, Proc. Natl. Acad.Sci. USA (1997) 94:12457-12461; Kambadur et al. Genome Res. (1997)7:910-915; and Schuelke et al. (2004) N Engl J Med, 350:2682-8]. Studieshave also shown that muscle wasting associated with HIV-infection inhumans is accompanied by increases in GDF8 protein expression[Gonzalez-Cadavid et al., PNAS (1998) 95:14938-43]. In addition, GDF8can modulate the production of muscle-specific enzymes (e.g., creatinekinase) and modulate myoblast cell proliferation [International PatentApplication Publication No. WO 00/43781]. The GDF8 propeptide cannoncovalently bind to the mature GDF8 domain dimer, inactivating itsbiological activity [Miyazono et al. (1988) J. Biol. Chem., 263:6407-6415; Wakefield et al. (1988) J. Biol. Chem., 263; 7646-7654; andBrown et al. (1990) Growth Factors, 3: 35-43]. Other proteins which bindto GDF8 or structurally related proteins and inhibit their biologicalactivity include follistatin, and potentially, follistatin-relatedproteins [Gamer et al. (1999) Dev. Biol., 208: 222-232].

GDF11, also known as BMP11, is a secreted protein that is expressed inthe tail bud, limb bud, maxillary and mandibular arches, and dorsal rootganglia during mouse development [McPherron et al. (1999) Nat. Genet.,22: 260-264; and Nakashima et al. (1999) Mech. Dev., 80: 185-189]. GDF11plays a unique role in patterning both mesodermal and neural tissues[Gamer et al. (1999) Dev Biol., 208:222-32]. GDF11 was shown to be anegative regulator of chondrogenesis and myogenesis in developing chicklimb [Gamer et al. (2001) Dev Biol., 229:407-20]. The expression ofGDF11 in muscle also suggests its role in regulating muscle growth in asimilar way to GDF8. In addition, the expression of GDF11 in brainsuggests that GDF11 may also possess activities that relate to thefunction of the nervous system. Interestingly, GDF11 was found toinhibit neurogenesis in the olfactory epithelium [Wu et al. (2003)Neuron., 37:197-207]. Hence, inhibitors GDF11 may have in vitro and invivo applications in the treatment of diseases such as muscle diseasesand neurodegenerative diseases (e.g., amyotrophic lateral sclerosis).

As described herein, comparative binding data demonstrated that anALK4:ActRIIB heterodimer has an altered binding profile (ligandselectivity) compared to either corresponding ActRIIB or ALK4homodimers. In particular, the ALK4:ActRIIB heterodimer displaysenhanced binding to activin B compared with either homodimer, andretains strong binding to activin A, GDF8, and GDF11 as observed withthe ActRIIB homodimer. However, the ALK4:ActRIIB heterodimer exhibitssubstantially reduced binding to BMP9, BMP10, and GDF3 compared to theActRIIB homodimer. In particular, BMP9 displays low or no observableaffinity for the ALK4:ActRIIB heterodimer, whereas this ligand bindsstrongly to ActRIIB homodimer.

These results therefore demonstrate that ALK4:ActRIIB heterodimers aremore selective antagonists of activin A, activin B, GDF8, and GDF11compared to ActRIIB homodimers. Accordingly, an ALK4:ActRIIB heterodimerwill be more useful than an ActRIIB homodimer in certain applicationswhere such selective antagonism is advantageous. Examples includetherapeutic applications where it is desirable to retain antagonism ofone or more of activin (e.g., activin A, activin B, activin AC, activinAB), GDF8, and GDF11 but minimize antagonism of one or more of BMP9,BMP10, and BMP6.

Moreover, ALK4:ActRIIB heterodimers, as described herein, exertbeneficial anabolic effects on skeletal muscle and bone as well ascatabolic effects on adipose tissue, very similar to those of an ActRIIBhomodimer. However, unlike ActRIIB homodimer, an ActRIIB:ALK4heterodimer exhibits only low-affinity or transient binding to BMP9 andBMP10 and so will have little to no concurrent inhibition on processesmediated by those ligands, such as angiogenesis. This novel selectivitywill be useful, for example, in treating patients in need of stimulatoryeffects on, e.g., muscle and bone, and inhibitory effects on fat, butnot in need of altered angiogenesis.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this disclosure and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification to provide additional guidanceto the practitioner in describing the compositions and methods of thedisclosure and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which it isused.

The terms “heteromer” or “heteromultimer” is a complex comprising atleast a first polypeptide chain and a second polypeptide chain, whereinthe second polypeptide chain differs in amino acid sequence from thefirst polypeptide chain by at least one amino acid residue. Theheteromer can comprise a “heterodimer” formed by the first and secondpolypeptide chains or can form higher order structures where one or morepolypeptide chains in addition to the first and second polypeptidechains are present. Exemplary structures for the heteromultimer includeheterodimers, heterotrimers, heterotetramers and further oligomericstructures. Heterodimers are designated herein as X:Y or equivalently asX-Y, where X represents a first polypeptide chain and Y represents asecond polypeptide chain. Higher-order heteromers and oligomericstructures are designated herein in a corresponding manner. In certainembodiments a heteromultimer is recombinant (e.g., one or morepolypeptide components may be a recombinant protein), isolated and/orpurified.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions. However, in common usage andin the instant application, the term “homologous,” when modified with anadverb such as “highly,” may refer to sequence similarity and may or maynot relate to a common evolutionary origin.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

“Percent (%) sequence identity” with respect to a reference polypeptide(or nucleotide) sequence is defined as the percentage of amino acidresidues (or nucleic acids) in a candidate sequence that are identicalto the amino acid residues (or nucleic acids) in the referencepolypeptide (nucleotide) sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid (nucleic acid) sequenceidentity values are generated using the sequence comparison computerprogram ALIGN-2. The ALIGN-2 sequence comparison computer program wasauthored by Genentech, Inc., and the source code has been filed withuser documentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available from Genentech, Inc., SouthSan Francisco, Calif., or may be compiled from the source code. TheALIGN-2 program should be compiled for use on a UNIX operating system,including digital UNIX V4.0D. All sequence comparison parameters are setby the ALIGN-2 program and do not vary.

“Agonize”, in all its grammatical forms, refers to the process ofactivating a protein and/or gene (e.g., by activating or amplifying thatprotein's gene expression or by inducing an inactive protein to enter anactive state) or increasing a protein's and/or gene's activity.

“Antagonize”, in all its grammatical forms, refers to the process ofinhibiting a protein and/or gene (e.g., by inhibiting or decreasing thatprotein's gene expression or by inducing an active protein to enter aninactive state) or decreasing a protein's and/or gene's activity.

The terms “about” and “approximately” as used in connection with anumerical value throughout the specification and the claims denotes aninterval of accuracy, familiar and acceptable to a person skilled in theart. In general, such interval of accuracy is ±10%. Alternatively, andparticularly in biological systems, the terms “about” and“approximately” may mean values that are within an order of magnitude,preferably ≤5-fold and more preferably ≤2-fold of a given value.

Numeric ranges disclosed herein are inclusive of the numbers definingthe ranges.

The terms “a” and “an” include plural referents unless the context inwhich the term is used clearly dictates otherwise. The terms “a” (or“an”), as well as the terms “one or more,” and “at least one” can beused interchangeably herein. Furthermore, “and/or” where used herein isto be taken as specific disclosure of each of the two or more specifiedfeatures or components with or without the other. Thus, the term“and/or” as used in a phrase such as “A and/or B” herein is intended toinclude “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, theterm “and/or” as used in a phrase such as “A, B, and/or C” is intendedto encompass each of the following aspects: A, B, and C; A, B, or C; Aor C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone);and C (alone).

2. ALK4:ActRIIB Heteromultimers

In certain aspects, the present disclosure relates to heteromultimerscomprising one or more ALK4 receptor polypeptides (e.g., SEQ ID NOs: 9,10, 19, 20, 42, 44, 47, 48, 74, and 76) and one or more ActRIIB receptorpolypeptides (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 39, 41, 45, 46, 70,72, 78, and 80) which are generally referred to herein as “ALK4:ActRIIBheteromultimer complexes” or “ALK4:ActRIIB heteromultimers”. Preferably,ALK4:ActRIIB heteromultimers of the disclosure are soluble, for example,a heteromultimer may comprises a soluble portion (domain) of an ALK4receptor and a soluble portion (domain) of an ActRIIB receptor. Ingeneral, the extracellular domains of ALK4 and ActRIIB correspond to asoluble portion of these receptors. Therefore, in some embodiments,heteromultimers of the disclosure comprise an extracellular domain of anALK4 receptor and an extracellular domain of an ActRIIB receptor.Example extracellular domains ALK4 and ActRIIB receptors are disclosedherein and such sequences, as well as fragments, functional variants,and modified forms thereof, may be used in accordance with theinventions of the disclosure (e.g., ALK4:ActRIIB heteromultimercompositions and uses thereof).

ALK4:ActRIIB heteromultimers of the disclosure include, e.g.,heterodimers, heterotrimers, heterotetramers and higher order oligomericstructures. See, e.g., FIG. 6. In certain preferred embodiments,heteromultimers of the disclosure are ALK4:ActRIIB heterodimers.

Preferably, ALK4:ActRIIB heteromultimers of the disclosure bind to oneor more TGF-beta superfamily ligands. In some embodiments, ALK4:ActRIIBheteromultimers may bind to one or more of activin (e.g., activin A,activin B, activin C, activin E, activin AC, activin AB, activin BC,activin AE, and activin BE), GDF8, GDF11, BMP6, GDF3, and BMP10. In someembodiments, ALK4:ActRIIB heteromultimers bind to activin A. In someembodiments, ALK4:ActRIIB heteromultimers bind to activin B. In someembodiments, ALK4:ActRIIB heteromultimers bind to activin C. In someembodiments, ALK4:ActRIIB heteromultimers bind to activin E. In someembodiments, ALK4:ActRIIB heteromultimers bind to activin AB. In someembodiments, ALK4:ActRIIB heteromultimers bind to activin AC. In someembodiments, ALK4:ActRIIB heteromultimers bind to activin AE. In someembodiments, ALK4:ActRIIB heteromultimers bind to activin BC. In someembodiments, ALK4:ActRIIB heteromultimers bind to activin BE. In someembodiments, ALK4:ActRIIB heteromultimers bind to GDF11. In someembodiments, ALK4:ActRIIB heteromultimers bind to GDF8. In someembodiments, ALK4:ActRIIB heteromultimers bind to BMP6. In someembodiments, ALK4:ActRIIB heteromultimers bind to GDF3. In someembodiments, ALK4:ActRIIB heteromultimers bind to BMP10. In someembodiments, ALK4:ActRIIB heteromultimers do not bind to, or no notsubstantially bind to BMP9 (e.g., have indeterminate K_(a) or K_(d) dueto the transient nature of the interaction between BMP9 and anALK4:ActRIIB heteromultimer). In some embodiments, ALK4:ActRIIBheteromultimers binds with stronger affinity to activin B compared to acorresponding ActRIIB homomultimer. In some embodiments, ALK4:ActRIIBheteromultimers binds with weaker affinity to GDF3 compared to acorresponding ActRIIB homomultimer. In some embodiments, ALK4:ActRIIBheteromultimers binds with weaker affinity to BMP9 compared to acorresponding ActRIIB homomultimer. In some embodiments, ALK4:ActRIIBheteromultimers binds with weaker affinity to BMP10 compared to acorresponding ActRIIB homomultimer. Optionally, ALK4:ActRIIBheteromultimers may further bind to one or more of BMP2, BMP2/7, BMP3,BMP4, BMP4/7, BMP5, BMP7, BMP8a, BMP8b, GDF5, GDF6/BMP13, GDF7,GDF9b/BMP15, GDF15/MIC1, TGF-β1, TGF-β2, TGF-β3, nodal, glialcell-derived neurotrophic factor (GDNF), neurturin, artemin, persephin,MIS, and Lefty.

In some embodiments, ALK4:ActRIIB heteromultimers may be used to inhibit(antagonize) signaling (e.g., Smad 2/3 and/or Smad 1/5/8 signaling)mediated by one or more TGFβ superfamily ligands. In particular,ALK4:ActRIIB heteromultimers of the disclosure may be used to inhibitintracellular signaling by one or more TGFβ superfamily ligands in, forexample, a cell-based assay such as those described herein. For example,ALK4:ActRIIB heteromultimers may inhibit signaling mediated by one ormore of activin (e.g., activin A, activin B, activin C, activin E,activin AC, activin AB, activin BC, activin AE, and activin BE), GDF8,GDF11, BMP6, GDF3, and BMP10 in a cell-based assay. In some embodiments,ALK4:ActRIIB heteromultimers may inhibit activin A signaling in acell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers mayinhibit activin B signaling in a cell-based assay. In some embodiments,ALK4:ActRIIB heteromultimers may inhibit activin C signaling in acell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers mayinhibit activin D signaling in a cell-based assay. In some embodiments,ALK4:ActRIIB heteromultimers may inhibit activin E signaling in acell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers mayinhibit activin AB signaling in a cell-based assay. In some embodiments,ALK4:ActRIIB heteromultimers may inhibit activin AC signaling in acell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers mayinhibit activin BC signaling in a cell-based assay. In some embodiments,ALK4:ActRIIB heteromultimers may inhibit activin AE signaling in acell-based assay. In some embodiments, ALK4:ActRIIB heteromultimers mayinhibit activin BE signaling in a cell-based assay. In some embodiments,ALK4:ActRIIB heteromultimers may inhibit GDF11 signaling in a cell-basedassay. In some embodiments, ALK4:ActRIIB heteromultimers may inhibitGDF8 signaling in a cell-based assay. In some embodiments, ALK4:ActRIIBheteromultimers may inhibit BMP6 signaling in a cell-based assay. Insome embodiments, ALK4:ActRIIB heteromultimers may inhibit GDF3signaling in a cell-based assay. In some embodiments, ALK4:ActRIIBheteromultimers may inhibit BMP9 signaling in a cell-based assay. Insome embodiments, ALK4:ActRIIB heteromultimers do not inhibit, or do notsubstantially inhibit BMP9 signaling in a cell-based assay. In someembodiments, ALK4:ActRIIB heteromultimers are stronger inhibitors ofactivin B signaling in a cell-based assay compared to a correspondingActRIIB homomultimer. In some embodiments, ALK4:ActRIIB heteromultimersare weaker inhibitors of BMP10 signaling in a cell-based assay comparedto a corresponding ActRIIB homomultimer. In some embodiments,ALK4:ActRIIB heteromultimers are stronger inhibitors of GDF3 signalingin a cell-based assay compared to a corresponding ActRIIB homomultimer.In some embodiments, ALK4:ActRIIB heteromultimers are strongerinhibitors of BMP9 signaling in a cell-based assay compared to acorresponding ActRIIB homomultimer. Optionally, ALK4:ActRIIBheteromultimers may further inhibit intracellular signaling by one ormore of BMP2, BMP2/7, BMP3, BMP4, BMP4/7, BMP5, BMP7, BMP8a, BMP8b,GDF5, GDF6/BMP13, GDF7, GDF9b/BMP15, GDF15/MIC1, TGF-β1, TGF-β2, TGF-β3,nodal, glial cell-derived neurotrophic factor (GDNF), neurturin,artemin, persephin, MIS, and Lefty in a cell-based assay.

As used herein, the term “ActRIIB” refers to a family of activinreceptor type IIB (ActRIIB) proteins from any species and variantsderived from such ActRIIB proteins by mutagenesis or other modification.Reference to ActRIIB herein is understood to be a reference to any oneof the currently identified forms. Members of the ActRIIB family aregenerally transmembrane proteins, composed of a ligand-bindingextracellular domain comprising a cysteine-rich region, a transmembranedomain, and a cytoplasmic domain with predicted serine/threonine kinaseactivity.

The term “ActRIIB polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ActRIIB family member as well asany variants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. Examples of suchvariant ActRIIB polypeptides are provided throughout the presentdisclosure as well as in International Patent Application PublicationNos. WO 2006/012627, WO 2008/097541, and WO 2010/151426, which areincorporated herein by reference in their entirety. Numbering of aminoacids for all ActRIIB-related polypeptides described herein is based onthe numbering of the human ActRIIB precursor protein sequence providedbelow (SEQ ID NO: 1), unless specifically designated otherwise.

The human ActRIIB precursor protein sequence is as follows:

(SEQ ID NO: 1)   1 MTAPWVALAL LWGSLCAGS G RGEAETRECI YYNANWELER T NQSGLERCE  51 GEQDKRLHCY ASWR N SSGTI ELVKKGCWLD DFNCYDRQEC VATEENPQVY101 FCCCEGNFCN ERFTHLPEAG GPEVTYEPPP TAPTLLTVLA YSLLPIGGLS 151LIVLLAFWMY RHRKPPYGHV DIHEDPGPPP PSPLVGLKPL QLLEIKARGR 201FGCVWKAQLM NDFVAVKIFP LQDKQSWQSE REIFSTPGMK HENLLQFIAA 251EKRGSNLEVE LWLITAFHDK GSLTDYLKGN IITWNELCHV AETMSRGLSY 301LHEDVPWCRG EGHKPSIAHR DFKSKNVLLK SDLTAVLADF GLAVRFEPGK 351PPGDTHGQVG TRRYMAPEVL EGAINFQRDA FLRIDMYAMG LVLWELVSRC 401KAADGPVDEY MLPFEEEIGQ HPSLEELQEV VVHKKMRPTI KDHWLKHPGL 451AQLCVTIEEC WDHDAEARLS AGCVEERVSL IRRSVNGTTS DCLVSLVTSV 501 TNVDLPPKES SI

The signal peptide is indicated with a single underline; theextracellular domain is indicated in bold font; and the potential,endogenous N-linked glycosylation sites are indicated with a doubleunderline.

The processed extracellular ActRIIB polypeptide sequence is as follows:

(SEQ ID NO: 2) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLP EAGGPEVTYEPPPTAPT

In some embodiments, the protein may be produced with an “SGR . . . ”sequence at the N-terminus. The C-terminal “tail” of the extracellulardomain is indicated by a single underline. The sequence with the “tail”deleted (a 415 sequence) is as follows:

(SEQ ID NO: 3) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLP EA

A form of ActRIIB with an alanine at position 64 of SEQ ID NO: 1 (A64)is also reported in the literature See, e.g., Hilden et al. (1994)Blood, 83(8): 2163-2170. Applicants have ascertained that an ActRIIB-Fcfusion protein comprising an extracellular domain of ActRIIB with theA64 substitution has a relatively low affinity for activin and GDF11. Bycontrast, the same ActRIIB-Fc fusion protein with an arginine atposition 64 (R64) has an affinity for activin and GDF11 in the lownanomolar to high picomolar range. Therefore, sequences with an R64 areused as the “wild-type” reference sequence for human ActRIIB in thisdisclosure.

The form of ActRIIB with an alanine at position 64 is as follows:

(SEQ ID NO: 4) 1 MTAPWVALAL LWGSLCAGS G RGEAETRECI YYNANWELER TNQSGLERCE51 GEQDKRLHCY ASWANSSGTI ELVKKGCWLD DFNCYDRQEC VATEENPQVY 101 FCCCEGNFCNERFTHLPEAG GPEVTYEPPP TAPTLLTVLA YSLLPIGGLS 151 LIVLLAFWMY RHRKPPYGHVDIHEDPGPPP PSPLVGLKPL QLLEIKARGR 201 FGCVWKAQLM NDFVAVKIFP LQDKQSWQSEREIFSTPGMK HENLLQFIAA 251 EKRGSNLEVE LWLITAFHDK GSLTDYLKGN IITWNELCHVAETMSRGLSY 301 LHEDVPWCRG EGHKPSIAHR DFKSKNVLLK SDLTAVLADF GLAVRFEPGK351 PPGDTHGQVG TRRYMAPEVL EGAINFQRDA FLRIDMYAMG LVLWELVSRC 401KAADGPVDEY MLPFEEEIGQ HPSLEELQEV VVHKKMRPTI KDHWLKHPGL 451 AQLCVTIEECWDHDAEARLS AGCVEERVSL IRRSVNGTTS DCLVSLVTSV 501 TNVDLPPKES SI

The signal peptide is indicated by single underline and theextracellular domain is indicated by bold font.

The processed extracellular ActRIIB polypeptide sequence of thealternative A64 form is as follows:

(SEQ ID NO: 5) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA GGPEVTYEPPPTAPT

In some embodiments, the protein may be produced with an “SGR . . . ”sequence at the N-terminus. The C-terminal “tail” of the extracellulardomain is indicated by single underline. The sequence with the “tail”deleted (a 415 sequence) is as follows:

(SEQ ID NO: 6) GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWANSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEA

A nucleic acid sequence encoding the human ActRIIB precursor protein isshown below (SEQ ID NO: 7), representing nucleotides 25-1560 of GenbankReference Sequence NM_001106.3, which encode amino acids 1-513 of theActRIIB precursor. The sequence as shown provides an arginine atposition 64 and may be modified to provide an alanine instead. Thesignal sequence is underlined.

(SEQ ID NO: 7) 1 ATGACGGCGC CCTGGGTGGC CCTCGCCCTC CTCTGGGGAT CGCTGTGCGC51 CGGCTCTGGG CGTGGGGAGG CTGAGACACG GGAGTGCATC TACTACAACG 101 CCAACTGGGAGCTGGAGCGC ACCAACCAGA GCGGCCTGGA GCGCTGCGAA 151 GGCGAGCAGG ACAAGCGGCTGCACTGCTAC GCCTCCTGGC GCAACAGCTC 201 TGGCACCATC GAGCTCGTGA AGAAGGGCTGCTGGCTAGAT GACTTCAACT 251 GCTACGATAG GCAGGAGTGT GTGGCCACTG AGGAGAACCCCCAGGTGTAC 301 TTCTGCTGCT GTGAAGGCAA CTTCTGCAAC GAACGCTTCA CTCATTTGCC351 AGAGGCTGGG GGCCCGGAAG TCACGTACGA GCCACCCCCG ACAGCCCCCA 401CCCTGCTCAC GGTGCTGGCC TACTCACTGC TGCCCATCGG GGGCCTTTCC 451 CTCATCGTCCTGCTGGCCTT TTGGATGTAC CGGCATCGCA AGCCCCCCTA 501 CGGTCATGTG GACATCCATGAGGACCCTGG GCCTCCACCA CCATCCCCTC 551 TGGTGGGCCT GAAGCCACTG CAGCTGCTGGAGATCAAGGC TCGGGGGCGC 601 TTTGGCTGTG TCTGGAAGGC CCAGCTCATG AATGACTTTGTAGCTGTCAA 651 GATCTTCCCA CTCCAGGACA AGCAGTCGTG GCAGAGTGAA CGGGAGATCT701 TCAGCACACC TGGCATGAAG CACGAGAACC TGCTACAGTT CATTGCTGCC 751GAGAAGCGAG GCTCCAACCT CGAAGTAGAG CTGTGGCTCA TCACGGCCTT 801 CCATGACAAGGGCTCCCTCA CGGATTACCT CAAGGGGAAC ATCATCACAT 851 GGAACGAACT GTGTCATGTAGCAGAGACGA TGTCACGAGG CCTCTCATAC 901 CTGCATGAGG ATGTGCCCTG GTGCCGTGGCGAGGGCCACA AGCCGTCTAT 951 TGCCCACAGG GACTTTAAAA GTAAGAATGT ATTGCTGAAGAGCGACCTCA 1001 CAGCCGTGCT GGCTGACTTT GGCTTGGCTG TTCGATTTGA GCCAGGGAAA1051 CCTCCAGGGG ACACCCACGG ACAGGTAGGC ACGAGACGGT ACATGGCTCC 1101TGAGGTGCTC GAGGGAGCCA TCAACTTCCA GAGAGATGCC TTCCTGCGCA 1151 TTGACATGTATGCCATGGGG TTGGTGCTGT GGGAGCTTGT GTCTCGCTGC 1201 AAGGCTGCAG ACGGACCCGTGGATGAGTAC ATGCTGCCCT TTGAGGAAGA 1251 GATTGGCCAG CACCCTTCGT TGGAGGAGCTGCAGGAGGTG GTGGTGCACA 1301 AGAAGATGAG GCCCACCATT AAAGATCACT GGTTGAAACACCCGGGCCTG 1351 GCCCAGCTTT GTGTGACCAT CGAGGAGTGC TGGGACCATG ATGCAGAGGC1401 TCGCTTGTCC GCGGGCTGTG TGGAGGAGCG GGTGTCCCTG ATTCGGAGGT 1451CGGTCAACGG CACTACCTCG GACTGTCTCG TTTCCCTGGT GACCTCTGTC 1501 ACCAATGTGGACCTGCCCCC TAAAGAGTCA AGCATC

A nucleic acid sequence encoding processed extracellular human ActRIIBpolypeptide is as follows (SEQ ID NO: 8). The sequence as shown providesan arginine at position 64, and may be modified to provide an alanineinstead.

(SEQ ID NO: 8) 1 GGGCGTGGGG AGGCTGAGAC ACGGGAGTGC ATCTACTACA ACGCCAACTG51 GGAGCTGGAG CGCACCAACC AGAGCGGCCT GGAGCGCTGC GAAGGCGAGC 101 AGGACAAGCGGCTGCACTGC TACGCCTCCT GGCGCAACAG CTCTGGCACC 151 ATCGAGCTCG TGAAGAAGGGCTGCTGGCTA GATGACTTCA ACTGCTACGA 201 TAGGCAGGAG TGTGTGGCCA CTGAGGAGAACCCCCAGGTG TACTTCTGCT 251 GCTGTGAAGG CAACTTCTGC AACGAACGCT TCACTCATTTGCCAGAGGCT 301 GGGGGCCCGG AAGTCACGTA CGAGCCACCC CCGACAGCCC CCACC

An alignment of the amino acid sequences of human ActRIIB extracellulardomain and human ActRIIA extracellular domain are illustrated in FIG. 1.This alignment indicates amino acid residues within both receptors thatare believed to directly contact ActRII ligands. For example, thecomposite ActRII structures indicated that the ActRIIB-ligand bindingpocket is defined, in part, by residues Y31, N33, N35, L38 through T41,E47, E50, Q53 through K55, L57, H58, Y60, S62, K74, W78 through N83,Y85, R87, A92, and E94 through F101. At these positions, it is expectedthat conservative mutations will be tolerated.

In addition, ActRIIB is well-conserved among vertebrates, with largestretches of the extracellular domain completely conserved. For example,FIG. 2 depicts a multi-sequence alignment of a human ActRIIBextracellular domain compared to various ActRIIB orthologs. Many of theligands that bind to ActRIIB are also highly conserved. Accordingly,from these alignments, it is possible to predict key amino acidpositions within the ligand-binding domain that are important for normalActRIIB-ligand binding activities as well as to predict amino acidpositions that are likely to be tolerant of substitution withoutsignificantly altering normal ActRIIB-ligand binding activities.Therefore, an active, human ActRIIB variant polypeptide useful inaccordance with the presently disclosed methods may include one or moreamino acids at corresponding positions from the sequence of anothervertebrate ActRIIB, or may include a residue that is similar to that inthe human or other vertebrate sequences. Without meaning to be limiting,the following examples illustrate this approach to defining an activeActRIIB variant. L46 in the human extracellular domain (SEQ ID NO: 53)is a valine in Xenopus ActRIIB (SEQ ID NO: 55), and so this position maybe altered, and optionally may be altered to another hydrophobicresidue, such as V, I or F, or a non-polar residue such as A. E52 in thehuman extracellular domain is a K in Xenopus, indicating that this sitemay be tolerant of a wide variety of changes, including polar residues,such as E, D, K, R, H, S, T, P, G, Y and probably A. T93 in the humanextracellular domain is a K in Xenopus, indicating that a widestructural variation is tolerated at this position, with polar residuesfavored, such as S, K, R, E, D, H, G, P, G and Y. F108 in the humanextracellular domain is a Y in Xenopus, and therefore Y or otherhydrophobic group, such as I, V or L should be tolerated. E111 in thehuman extracellular domain is K in Xenopus, indicating that chargedresidues will be tolerated at this position, including D, R, K and H, aswell as Q and N. R112 in the human extracellular domain is K in Xenopus,indicating that basic residues are tolerated at this position, includingR and H. A at position 119 in the human extracellular domain isrelatively poorly conserved, and appears as P in rodents and V inXenopus, thus essentially any amino acid should be tolerated at thisposition.

Moreover, ActRII proteins have been characterized in the art in terms ofstructural and functional characteristics, particularly with respect toligand binding [Attisano et al. (1992) Cell 68(1):97-108; Greenwald etal. (1999) Nature Structural Biology 6(1): 18-22; Allendorph et al.(2006) PNAS 103(20: 7643-7648; Thompson et al. (2003) The EMBO Journal22(7): 1555-1566; as well as U.S. Pat. Nos. 7,709,605, 7,612,041, and7,842,663]. In addition to the teachings herein, these referencesprovide amply guidance for how to generate ActRIIB variants that retainone or more normal activities (e.g., ligand-binding activity).

For example, a defining structural motif known as a three-finger toxinfold is important for ligand binding by type I and type II receptors andis formed by conserved cysteine residues located at varying positionswithin the extracellular domain of each monomeric receptor [Greenwald etal. (1999) Nat Struct Biol 6:18-22; and Hinck (2012) FEBS Lett586:1860-1870]. Accordingly, the core ligand-binding domains of humanActRIIB, as demarcated by the outermost of these conserved cysteines,corresponds to positions 29-109 of SEQ ID NO: 1 (ActRIIB precursor).Thus, the structurally less-ordered amino acids flanking thesecysteine-demarcated core sequences can be truncated by about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, or 28 residues at the N-terminus and/or by about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 residues at the C-terminus without necessarily alteringligand binding. Exemplary ActRIIB extracellular domains for N-terminaland/or C-terminal truncation include SEQ ID NOs: 2, 3, 5, and 6.

Attisano et al. showed that a deletion of the proline knot at theC-terminus of the extracellular domain of ActRIIB reduced the affinityof the receptor for activin. An ActRIIB-Fc fusion protein containingamino acids 20-119 of present SEQ ID NO: 1, “ActRIIB(20-119)-Fc”, hasreduced binding to GDF11 and activin relative to an ActRIIB(20-134)-Fc,which includes the proline knot region and the complete juxtamembranedomain (see, e.g., U.S. Pat. No. 7,842,663). However, anActRIIB(20-129)-Fc protein retains similar, but somewhat reducedactivity, relative to the wild-type, even though the proline knot regionis disrupted.

Thus, ActRIIB extracellular domains that stop at amino acid 134, 133,132, 131, 130 and 129 (with respect to SEQ ID NO: 1) are all expected tobe active, but constructs stopping at 134 or 133 may be most active.Similarly, mutations at any of residues 129-134 (with respect to SEQ IDNO: 1) are not expected to alter ligand-binding affinity by largemargins. In support of this, it is known in the art that mutations ofP129 and P130 (with respect to SEQ ID NO: 1) do not substantiallydecrease ligand binding. Therefore, an ActRIIB polypeptide of thepresent disclosure may end as early as amino acid 109 (the finalcysteine), however, forms ending at or between 109 and 119 (e.g., 109,110, 111, 112, 113, 114, 115, 116, 117, 118, or 119) are expected tohave reduced ligand binding. Amino acid 119 (with respect to present SEQID NO:1) is poorly conserved and so is readily altered or truncated.ActRIIB polypeptides ending at 128 (with respect to SEQ ID NO: 1) orlater should retain ligand-binding activity. ActRIIB polypeptides endingat or between 119 and 127 (e.g., 119, 120, 121, 122, 123, 124, 125, 126,or 127), with respect to SEQ ID NO: 1, will have an intermediate bindingability. Any of these forms may be desirable to use, depending on theclinical or experimental setting.

At the N-terminus of ActRIIB, it is expected that a protein beginning atamino acid 29 or before (with respect to SEQ ID NO: 1) will retainligand-binding activity. Amino acid 29 represents the initial cysteine.An alanine-to-asparagine mutation at position 24 (with respect to SEQ IDNO: 1) introduces an N-linked glycosylation sequence withoutsubstantially affecting ligand binding [U.S. Pat. No. 7,842,663]. Thisconfirms that mutations in the region between the signal cleavagepeptide and the cysteine cross-linked region, corresponding to aminoacids 20-29, are well tolerated. In particular, ActRIIB polypeptidesbeginning at position 20, 21, 22, 23, and 24 (with respect to SEQ IDNO: 1) should retain general ligand-biding activity, and ActRIIBpolypeptides beginning at positions 25, 26, 27, 28, and 29 (with respectto SEQ ID NO: 1) are also expected to retain ligand-biding activity. Ithas been demonstrated, e.g., U.S. Pat. No. 7,842,663, that,surprisingly, an ActRIIB construct beginning at 22, 23, 24, or 25 willhave the most activity.

Taken together, a general formula for an active portion (e.g.,ligand-binding portion) of ActRIIB comprises amino acids 29-109 of SEQID NO: 1. Therefore ActRIIB polypeptides may, for example, comprise,consist essentially of, or consist of an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of ActRIIBbeginning at a residue corresponding to any one of amino acids 20-29(e.g., beginning at any one of amino acids 20, 21, 22, 23, 24, 25, 26,27, 28, or 29) of SEQ ID NO: 1 and ending at a position corresponding toany one amino acids 109-134 (e.g., ending at any one of amino acids 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) of SEQ IDNO: 1. Other examples include polypeptides that begin at a position from20-29 (e.g., any one of positions 20, 21, 22, 23, 24, 25, 26, 27, 28, or29) or 21-29 (e.g., any one of positions 21, 22, 23, 24, 25, 26, 27, 28,or 29) of SEQ ID NO: 1 and end at a position from 119-134 (e.g., any oneof positions 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, or 134), 119-133 (e.g., any one of positions 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133),129-134 (e.g., any one of positions 129, 130, 131, 132, 133, or 134), or129-133 (e.g., any one of positions 129, 130, 131, 132, or 133) of SEQID NO: 1. Other examples include constructs that begin at a positionfrom 20-24 (e.g., any one of positions 20, 21, 22, 23, or 24), 21-24(e.g., any one of positions 21, 22, 23, or 24), or 22-25 (e.g., any oneof positions 22, 22, 23, or 25) of SEQ ID NO: 1 and end at a positionfrom 109-134 (e.g., any one of positions 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, or 134), 119-134 (e.g., any one of positions119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, or 134) or 129-134 (e.g., any one of positions 129, 130, 131, 132,133, or 134) of SEQ ID NO: 1. Variants within these ranges are alsocontemplated, particularly those having at least 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identity to the corresponding portion of SEQ ID NO: 1.

The variations described herein may be combined in various ways. In someembodiments, ActRIIB variants comprise no more than 1, 2, 5, 6, 7, 8, 9,10 or 15 conservative amino acid changes in the ligand-binding pocket,and zero, one, or more non-conservative alterations at positions 40, 53,55, 74, 79 and/or 82 in the ligand-binding pocket. Sites outside thebinding pocket, at which variability may be particularly well tolerated,include the amino and carboxy termini of the extracellular domain (asnoted above), and positions 42-46 and 65-73 (with respect to SEQ ID NO:1). An asparagine-to-alanine alteration at position 65 (N65A) actuallyimproves ligand binding in the A64 background, and is thus expected tohave no detrimental effect on ligand binding in the R64 background [U.S.Pat. No. 7,842,663]. This change probably eliminates glycosylation atN65 in the A64 background, thus demonstrating that a significant changein this region is likely to be tolerated. While an R64A change is poorlytolerated, R64K is well-tolerated, and thus another basic residue, suchas H may be tolerated at position 64 [U.S. Pat. No. 7,842,663].Additionally, the results of the mutagenesis program described in theart indicate that there are amino acid positions in ActRIIB that areoften beneficial to conserve. With respect to SEQ ID NO: 1, theseinclude position 80 (acidic or hydrophobic amino acid), position 78(hydrophobic, and particularly tryptophan), position 37 (acidic, andparticularly aspartic or glutamic acid), position 56 (basic amino acid),position 60 (hydrophobic amino acid, particularly phenylalanine ortyrosine). Thus, the disclosure provides a framework of amino acids thatmay be conserved in ActRIIB polypeptides. Other positions that may bedesirable to conserve are as follows: position 52 (acidic amino acid),position 55 (basic amino acid), position 81 (acidic), 98 (polar orcharged, particularly E, D, R or K), all with respect to SEQ ID NO: 1.

In certain embodiments, the disclosure relates to heteromultimers thatcomprise at least one ActRIIB polypeptide, which includes fragments,functional variants, and modified forms thereof. Preferably, ActRIIBpolypeptides for use in accordance with inventions of the disclosure aresoluble (e.g., an extracellular domain of ActRIIB). In other preferredembodiments, ActRIIB polypeptides for use in accordance with thedisclosure bind to one or more TGF-beta superfamily ligands. Therefore,in some embodiments, ActRIIB polypeptides for use in accordance with thedisclosure inhibit (antagonize) activity (e.g., inhibition of Smadsignaling) of one or more TGF-beta superfamily ligands. In someembodiments, heteromultimers of the disclosure comprise at least oneActRIIB polypeptide that comprise, consist essentially of, or consist ofan amino acid sequence that is at least 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a portion of ActRIIB beginning at a residue correspondingto amino acids 20-29 (e.g., beginning at any one of amino acids 20, 21,22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 1 and ending at aposition corresponding to amino acids 109-134 (e.g., ending at any oneof amino acids 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or134) of SEQ ID NO: 1. In certain preferred embodiments, heteromultimersof the disclosure comprise at least one ActRIIB polypeptide thatcomprises, consists, or consists essentially of an amino acid sequencethat is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acids 29-109of SEQ ID NO: 1 In other preferred embodiments, heteromultimer complexesof the disclosure comprise at least one ActRIIB polypeptide thatcomprises, consists of, or consists essentially of an amino acidsequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical aminoacids 25-131 of SEQ ID NO: 1. In some embodiments, heteromultimers ofthe disclosure comprise at least one ActRIIB polypeptide that is atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of anyone of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 39, 41, 45, or 46. In certainpreferred embodiments, heteromultimers of the disclosure comprise do notcomprise an ActRIIB polypeptide wherein the position corresponding toL79 of SEQ ID NO: 1 is an acidic amino acid (i.e., is not a naturallyoccurring D or E amino acid residue or artificial acidic amino acid).

In certain aspects, the present disclosure relates to protein complexesthat comprise an ALK4 polypeptide. As used herein, the term “ALK4”refers to a family of activin receptor-like kinase-4 proteins from anyspecies and variants derived from such ALK4 proteins by mutagenesis orother modification. Reference to ALK4 herein is understood to be areference to any one of the currently identified forms. Members of theALK4 family are generally transmembrane proteins, composed of aligand-binding extracellular domain with a cysteine-rich region, atransmembrane domain, and a cytoplasmic domain with predictedserine/threonine kinase activity.

The term “ALK4 polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ALK4 family member as well as anyvariants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. Numbering of aminoacids for all ALK4-related polypeptides described herein is based on thenumbering of the human ALK4 precursor protein sequence below (SEQ ID NO:9), unless specifically designated otherwise.

A human ALK4 precursor protein sequence (NCBI Ref Seq NP_0042931) is asfollows:

(SEQ ID NO: 9) 1 MAESAGASSF FPLVVLLLAG SGG SGPRGVQ ALLCACTSCL QANYTCETDGACMVSIFNLD 61 GMEHHVRTCI PKVELVPAGK PFYCLSSEDL RNTHCCYTDY CNRIDLRVPSGHLKEPEHPS 121 MWGPVELVGI IAGPVFLLFL IIIIVFLVIN YHQRVYHNRQ RLDMEDPSCEMCLSKDKTLQ 181 DLVYDLSTSG SGSGLPLFVQ RTVARTIVLQ EIIGKGRFGE VWRGRWRGGDVAVKIFSSRE 241 ERSWFREAEI YQTVMLRHEN ILGFIAADNK DNGTWTQLWL VSDYHEHGSLFDYLNRYTVT 301 IEGMIKLALS AASGLAHLHM EIVGTQGKPG IAHRDLKSKN ILVKKNGMCAIADLGLAVRH 361 DAVTDTIDIA PNQRVGTKRY MAPEVLDETI NMKHFDSFKC ADIYALGLVYWEIARRCNSG 421 GVHEEYQLPY YDLVPSDPSI EEMRKVVCDQ KLRPNIPNWW QSYEALRVMGKMMRECWYAN 481 GAARLTALRI KKTLSQLSVQ EDVKI

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

A processed extracellular human ALK4 polypeptide sequence is as follows:

(SEQ ID NO: 10) SGPRGVQALLCACTSCLQANYTCETDGACMVSIFNLDGMEHHVRTCIPKVELVPAGKPFYCLSSEDLRNTHCCYTDYCNRIDLRVPSGHLKEPEHPSMWG PVE

A nucleic acid sequence encoding the ALK4 precursor protein is shownbelow (SEQ ID NO: 11), corresponding to nucleotides 78-1592 of GenbankReference Sequence NM_004302.4. The signal sequence is underlined andthe extracellular domain is indicated in bold font.

(SEQ ID NO: 11) ATGGCGGAGTCGGCCGGAGCCTCCTCCTTCTTCCCCCTTGTTGTCCTCCTGCTCGCCGGCAGCGGCGGG TCCGGGCCCCGGGGGGTCCAGGCTCTGCTGTGTGCGTGCACCAGCTGCCTCCAGGCCAACTACACGTGTGAGACAGATGGGGCCTGCATGGTTTCCATTTTCAATCTGGATGGGATGGAGCACCATGTGCGCACCTGCATCCCCAAAGTGGAGCTGGTCCCTGCCGGGAAGCCCTTCTACTGCCTGAGCTCGGAGGACCTGCGCAACACCCACTGCTGCTACACTGACTACTGCAACAGGATCGACTTGAGGGTGCCCAGTGGTCACCTCAAGGAGCCTGAGCACCCGTCCATGTGGGGCCCGGTGGAGCTGGTAGGCATCATCGCCGGCCCGGTGTTCCTCCTGTTCCTCATCATCATCATTGTTTTCCTTGTCATTAACTATCATCAGCGTGTCTATCACAACCGCCAGAGACTGGACATGGAAGATCCCTCATGTGAGATGTGTCTCTCCAAAGACAAGACGCTCCAGGATCTTGTCTACGATCTCTCCACCTCAGGGTCTGGCTCAGGGTTACCCCTCTTTGTCCAGCGCACAGTGGCCCGAACCATCGTTTTACAAGAGATTATTGGCAAGGGTCGGTTTGGGGAAGTATGGCGGGGCCGCTGGAGGGGTGGTGATGTGGCTGTGAAAATATTCTCTTCTCGTGAAGAACGGTCTTGGTTCAGGGAAGCAGAGATATACCAGACGGTCATGCTGCGCCATGAAAACATCCTTGGATTTATTGCTGCTGACAATAAAGATAATGGCACCTGGACACAGCTGTGGCTTGTTTCTGACTATCATGAGCACGGGTCCCTGTTTGATTATCTGAACCGGTACACAGTGACAATTGAGGGGATGATTAAGCTGGCCTTGTCTGCTGCTAGTGGGCTGGCACACCTGCACATGGAGATCGTGGGCACCCAAGGGAAGCCTGGAATTGCTCATCGAGACTTAAAGTCAAAGAACATTCTGGTGAAGAAAAATGGCATGTGTGCCATAGCAGACCTGGGCCTGGCTGTCCGTCATGATGCAGTCACTGACACCATTGACATTGCCCCGAATCAGAGGGTGGGGACCAAACGATACATGGCCCCTGAAGTACTTGATGAAACCATTAATATGAAACACTTTGACTCCTTTAAATGTGCTGATATTTATGCCCTCGGGCTTGTATATTGGGAGATTGCTCGAAGATGCAATTCTGGAGGAGTCCATGAAGAATATCAGCTGCCATATTACGACTTAGTGCCCTCTGACCCTTCCATTGAGGAAATGCGAAAGGTTGTATGTGATCAGAAGCTGCGTCCCAACATCCCCAACTGGTGGCAGAGTTATGAGGCACTGCGGGTGATGGGGAAGATGATGCGAGAGTGTTGGTATGCCAACGGCGCAGCCCGCCTGACGGCCCTGCGCATCAAGAAGACCCTCTCCCAGCTCAGCGTGCAG GAAGACGTGAAGATC

A nucleic acid sequence encoding an extracellular ALK4 polypeptide is asfollows:

(SEQ ID NO: 12) TCCGGGCCCCGGGGGGTCCAGGCTCTGCTGTGTGCGTGCACCAGCTGCCTCCAGGCCAACTACACGTGTGAGACAGATGGGGCCTGCATGGTTTCCATTTTCAATCTGGATGGGATGGAGCACCATGTGCGCACCTGCATCCCCAAAGTGGAGCTGGTCCCTGCCGGGAAGCCCTTCTACTGCCTGAGCTCGGAGGACCTGCGCAACACCCACTGCTGCTACACTGACTACTGCAACAGGATCGACTTGAGGGTGCCCAGTGGTCACCTCAAGGAGCCTGAGCACCCGTCCATGTGGGGC CCGGTGGAG

An alternative isoform of human ALK4 precursor protein sequence, isoformC (NCBI Ref Seq NP_064733.3), is as follows:

(SEQ ID NO: 19) 1 MAESAGASSF FPLVVLLLAG SGG SGPRGVQ ALLCACTSCLQANYTCETDG ACMVSIFNLD 61 GMEHHVRTCI PKVELVPAGK PFYCLSSEDL RNTHCCYTDYCNRIDLRVPS GHLKEPEHPS 121 MWGPVELVGI IAGPVFLLFL IIIIVFLVIN YHQRVYHNRQRLDMEDPSCE MCLSKDKTLQ 181 DLVYDLSTSG SGSGLPLFVQ RTVARTIVLQ EIIGKGRFGEVWRGRWRGGD VAVKIFSSRE 241 ERSWFREAEI YQTVMLRHEN ILGFIAADNK ADCSFLTLPWEVVMVSAAPK LRSLRLQYKG 301 GRGRARFLFP LNNGTWTQLW LVSDYHEHGS LFDYLNRYTVTIEGMIKLAL SAASGLAHLH 361 MEIVGTQGKP GIAHRDLKSK NILVKKNGMC AIADLGLAVRHDAVTDTIDI APNQRVGTKR 421 YMAPEVLDET INMKHFDSFK CADIYALGLV YWEIARRCNSGGVHEEYQLP YYDLVPSDPS 481 IEEMRKVVCD QKLRPNIPNW WQSYEALRVM GKMMRECWYANGAARLTALR IKKTLSQLSV 541 QEDVKI

The signal peptide is indicated by a single underline and theextracellular domain is indicated in bold font.

A processed extracellular ALK4 polypeptide sequence (isoform C) is asfollows:

(SEQ ID NO: 20) SGPRGVQALLCACTSCLQANYTCETDGACMVSIFNLDGMEHHVRTCIPKVELVPAGKPFYCLSSEDLRNTHCCYTDYCNRIDLRVPSGHLKEPEHPSMWG PVE

A nucleic acid sequence encoding the ALK4 precursor protein (isoform C)is shown below (SEQ ID NO: 21), corresponding to nucleotides 78-1715 ofGenbank Reference Sequence NM_020328.3. The signal sequence isunderlined and the extracellular domain is indicated in bold font.

(SEQ ID NO: 21) ATGGCGGAGTCGGCCGGAGCCTCCTCCTTCTTCCCCCTTGTTGTCCTCCTGCTCGCCGGCAGCGGCGGG TCCGGGCCCCGGGGGGTCCAGGCTCTGCTGTGTGCGTGCACCAGCTGCCTCCAGGCCAACTACACGTGTGAGACAGATGGGGCCTGCATGGTTTCCATTTTCAATCTGGATGGGATGGAGCACCATGTGCGCACCTGCATCCCCAAAGTGGAGCTGGTCCCTGCCGGGAAGCCCTTCTACTGCCTGAGCTCGGAGGACCTGCGCAACACCCACTGCTGCTACACTGACTACTGCAACAGGATCGACTTGAGGGTGCCCAGTGGTCACCTCAAGGAGCCTGAGCACCCGTCCATGTGGGGCCCGGTGGAGCTGGTAGGCATCATCGCCGGCCCGGTGTTCCTCCTGTTCCTCATCATCATCATTGTTTTCCTTGTCATTAACTATCATCAGCGTGTCTATCACAACCGCCAGAGACTGGACATGGAAGATCCCTCATGTGAGATGTGTCTCTCCAAAGACAAGACGCTCCAGGATCTTGTCTACGATCTCTCCACCTCAGGGTCTGGCTCAGGGTTACCCCTCTTTGTCCAGCGCACAGTGGCCCGAACCATCGTTTTACAAGAGATTATTGGCAAGGGTCGGTTTGGGGAAGTATGGCGGGGCCGCTGGAGGGGTGGTGATGTGGCTGTGAAAATATTCTCTTCTCGTGAAGAACGGTCTTGGTTCAGGGAAGCAGAGATATACCAGACGGTCATGCTGCGCCATGAAAACATCCTTGGATTTATTGCTGCTGACAATAAAGCAGACTGCTCATTCCTCACATTGCCATGGGAAGTTGTAATGGTCTCTGCTGCCCCCAAGCTGAGGAGCCTTAGACTCCAATACAAGGGAGGAAGGGGAAGAGCAAGATTTTTATTCCCACTGAATAATGGCACCTGGACACAGCTGTGGCTTGTTTCTGACTATCATGAGCACGGGTCCCTGTTTGATTATCTGAACCGGTACACAGTGACAATTGAGGGGATGATTAAGCTGGCCTTGTCTGCTGCTAGTGGGCTGGCACACCTGCACATGGAGATCGTGGGCACCCAAGGGAAGCCTGGAATTGCTCATCGAGACTTAAAGTCAAAGAACATTCTGGTGAAGAAAAATGGCATGTGTGCCATAGCAGACCTGGGCCTGGCTGTCCGTCATGATGCAGTCACTGACACCATTGACATTGCCCCGAATCAGAGGGTGGGGACCAAACGATACATGGCCCCTGAAGTACTTGATGAAACCATTAATATGAAACACTTTGACTCCTTTAAATGTGCTGATATTTATGCCCTCGGGCTTGTATATTGGGAGATTGCTCGAAGATGCAATTCTGGAGGAGTCCATGAAGAATATCAGCTGCCATATTACGACTTAGTGCCCTCTGACCCTTCCATTGAGGAAATGCGAAAGGTTGTATGTGATCAGAAGCTGCGTCCCAACATCCCCAACTGGTGGCAGAGTTATGAGGCACTGCGGGTGATGGGGAAGATGATGCGAGAGTGTTGGTATGCCAACGGCGCAGCCCGCCTGACGGCCCTGCGCATCAAGAAGACCCTCTCCCAGCTCAGCGTGCAGGAAGACGTGAAGATC

A nucleic acid sequence encoding an extracellular ALK4 polypeptide(isoform C) is as follows:

(SEQ ID NO: 22) TCCGGGCCCCGGGGGGTCCAGGCTCTGCTGTGTGCGTGCACCAGCTGCCTCCAGGCCAACTACACGTGTGAGACAGATGGGGCCTGCATGGTTTCCATTTTCAATCTGGATGGGATGGAGCACCATGTGCGCACCTGCATCCCCAAAGTGGAGCTGGTCCCTGCCGGGAAGCCCTTCTACTGCCTGAGCTCGGAGGACCTGCGCAACACCCACTGCTGCTACACTGACTACTGCAACAGGATCGACTTGAGGGTGCCCAGTGGTCACCTCAAGGAGCCTGAGCACCCGTCCATGTGGGGC CCGGTGGAG

In certain embodiments, the disclosure relates to heteromultimers thatcomprise at least one ALK4 polypeptide, which includes fragments,functional variants, and modified forms thereof. Preferably, ALK4polypeptides for use in accordance with inventions of the disclosure(e.g., heteromultimers comprising an ALK4 polypeptide and uses thereof)are soluble (e.g., an extracellular domain of ALK4). In other preferredembodiments, ALK4 polypeptides for use in accordance with the inventionsof the disclosure bind to and/or inhibit (antagonize) activity (e.g.,Smad signaling) of one or more TGF-beta superfamily ligands. In someembodiments, heteromultimers of the disclosure comprise at least oneALK4 polypeptide that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% identical to theamino acid sequence of SEQ ID NO: 9, 10, 19, 20, 42, 44, 47, 48, 74, and76. In some embodiments, heteromultimer complexes of the disclosureconsist or consist essentially of at least one ALK4 polypeptide that isat least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 97%, 98%, or 99% identical to the amino acid sequence of SEQID NO: 9, 10, 19, 20, 42, 44, 47, 48, 74, and 76.

ALK4 is well-conserved among vertebrates, with large stretches of theextracellular domain completely conserved. For example, FIG. 5 depicts amulti-sequence alignment of a human ALK4 extracellular domain comparedto various ALK4 orthologs. Many of the ligands that bind to ALK4 arealso highly conserved. Accordingly, from these alignments, it ispossible to predict key amino acid positions within the ligand-bindingdomain that are important for normal ALK4-ligand binding activities aswell as to predict amino acid positions that are likely to be tolerantto substitution without significantly altering normal ALK4-ligandbinding activities. Therefore, an active, human ALK4 variant polypeptideuseful in accordance with the presently disclosed methods may includeone or more amino acids at corresponding positions from the sequence ofanother vertebrate ALK4, or may include a residue that is similar tothat in the human or other vertebrate sequences. Without meaning to belimiting, the following examples illustrate this approach to defining anactive ALK4 variant. V6 in the human ALK4 extracellular domain (SEQ IDNO: 59) is isoleucine in Mus muculus ALK4 (SEQ ID NO: 63), and so theposition may be altered, and optionally may be altered to anotherhydrophobic residue such as L, I, or F, or a non-polar residue such asA, as is observed in Gallus gallus ALK4 (SEQ ID NO: 62). E40 in thehuman extracellular domain is K in Gallus gallus ALK4, indicating thatthis site may be tolerant of a wide variety of changes, including polarresidues, such as E, D, K, R, H, S, T, P, G, Y, and probably a non-polarresidue such as A. S15 in the human extracellular domain is D in Gallusgallus ALK4, indicating that a wide structural variation is tolerated atthis position, with polar residues favored, such as S, T, R, E, K, H, G,P, G and Y. E40 in the human extracellular domain is K in Gallus gallusALK4, indicating that charged residues will be tolerated at thisposition, including D, R, K, H, as well as Q and N. R80 in the humanextracellular domain is K in Condylura cristata ALK4 (SEQ ID NO: 60),indicating that basic residues are tolerated at this position, includingR, K, and H. Y77 in the human extracellular domain is F in Sus scrofaALK4 (SEQ ID NO: 64), indicating that aromatic residues are tolerated atthis position, including F, W, and Y. P93 in the human extracellulardomain is relatively poorly conserved, appearing as S in Erinaceuseuropaeus ALK4 (SEQ ID NO: 61) and N in Gallus gallus ALK4, thusessentially any amino acid should be tolerated at this position.

Moreover, ALK4 proteins have been characterized in the art in terms ofstructural and functional characteristics, particularly with respect toligand binding [e.g., Harrison et al. (2003) J Biol Chem278(23):21129-21135; Romano et al. (2012) J Mol Model 18(8):3617-3625;and Calvanese et al. (2009) 15(3):175-183]. In addition to the teachingsherein, these references provide amply guidance for how to generate ALK4variants that retain one or more normal activities (e.g., ligand-bindingactivity).

For example, a defining structural motif known as a three-finger toxinfold is important for ligand binding by type I and type II receptors andis formed by conserved cysteine residues located at varying positionswithin the extracellular domain of each monomeric receptor [Greenwald etal. (1999) Nat Struct Biol 6:18-22; and Hinck (2012) FEBS Lett586:1860-1870]. Accordingly, the core ligand-binding domains of humanALK4, as demarcated by the outermost of these conserved cysteines,corresponds to positions 34-101 of SEQ ID NO: 9 (ALK4 precursor). Thus,the structurally less-ordered amino acids flanking thesecysteine-demarcated core sequences can be truncated by 1, 2, 3, 4, 5, 6,7, 8, 9, or 10, residues at the N-terminus or 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26residues at the C-terminus without necessarily altering ligand binding.Exemplary ALK4 extracellular domains for N-terminal and/or C-terminaltruncation include SEQ ID NOs: 10 and 20.

Accordingly, a general formula for an active portion (e.g., aligand-binding portion) of ALK4 comprises amino acids 34-101. ThereforeALK4 polypeptides may, for example, comprise, consists essentially of,or consists of an amino acid sequence that is at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to a portion of ALK4 beginning at a residuecorresponding to any one of amino acids 24-34 (e.g., beginning at anyone of amino acids 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34) of SEQID NO: 1 and ending at a position corresponding to any one amino acids101-126 (e.g., ending at any one of amino acids 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, or 126) of SEQ ID NO: 9. Other examplesinclude constructs that begin at a position from 24-34 (e.g., any one ofpositions 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34), 25-34 (e.g.,any one of positions 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34), or26-34 (e.g., any one of positions 26, 27, 28, 29, 30, 31, 32, 33, or 34)of SEQ ID NO: 9 and end at a position from 101-126 (e.g., any one ofpositions 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, or126), 102-126 (e.g., any one of positions 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, or 126), 101-125 (e.g., any one of positions 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, or 125), 101-124 (e.g., anyone of positions 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, or 124),101-121 (e.g., any one of positions 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, or121), 111-126 (e.g., any one of positions 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, or 126), 111-125 (e.g., anyone of positions 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, or 125), 111-124 (e.g., any one of positions 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, or 124), 121-126(e.g., any one of positions 121, 122, 123, 124, 125, or 126), 121-125(e.g., any one of positions 121, 122, 123, 124, or 125), 121-124 (e.g.,any one of positions 121, 122, 123, or 124), or 124-126 (e.g., any oneof positions 124, 125, or 126) of SEQ ID NO: 9. Variants within theseranges are also contemplated, particularly those having at least 70%,75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identity to the corresponding portion of SEQ IDNO: 9.

The variations described herein may be combined in various ways. In someembodiments, ALK4 variants comprise no more than 1, 2, 5, 6, 7, 8, 9, 10or 15 conservative amino acid changes in the ligand-binding pocket.Sites outside the binding pocket, at which variability may beparticularly well tolerated, include the amino and carboxy termini ofthe extracellular domain (as noted above),

In some embodiments, the present disclosure contemplates makingfunctional variants by modifying the structure of an ALK4 polypeptideand/or an ActRIIB polypeptide. Variants can be produced by amino acidsubstitution, deletion, addition, or combinations thereof. For instance,it is reasonable to expect that an isolated replacement of a leucinewith an isoleucine or valine, an aspartate with a glutamate, a threoninewith a serine, or a similar replacement of an amino acid with astructurally related amino acid (e.g., conservative mutations) will nothave a major effect on the biological activity of the resultingmolecule. Conservative replacements are those that take place within afamily of amino acids that are related in their side chains. Whether achange in the amino acid sequence of a polypeptide of the disclosureresults in a functional homolog can be readily determined by assessingthe ability of the variant polypeptide to produce a response in cells ina fashion similar to the wild-type polypeptide, or to bind to one ormore TGF-beta superfamily ligands including, for example, BMP2, BMP2/7,BMP3, BMP4, BMP4/7, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF3,GDF5, GDF6/BMP13, GDF7, GDF8, GDF9b/BMP15, GDF11/BMP11, GDF15/MIC1,TGF-β1, TGF-β2, TGF-β3, activin A, activin B, activin C, activin E,activin AB, activin AC, activin BC, activin AE, activin BE, nodal, glialcell-derived neurotrophic factor (GDNF), neurturin, artemin, persephin,MIS, and Lefty.

In some embodiments, the present disclosure contemplates makingfunctional variants by modifying the structure of an ALK4 and/or ActRIIBpolypeptide for such purposes as enhancing therapeutic efficacy orstability (e.g., increase shelf-life and/or resistance to proteolyticdegradation).

In some embodiments, the present disclosure contemplates specificmutations of an ALK4 polypeptide and/or an ActRIIB polypeptide so as toalter the glycosylation of the polypeptide. Such mutations may beselected so as to introduce or eliminate one or more glycosylationsites, such as O-linked or N-linked glycosylation sites.Asparagine-linked glycosylation recognition sites generally comprise atripeptide sequence, asparagine-X-threonine or asparagine-X-serine(where “X” is any amino acid) which is specifically recognized byappropriate cellular glycosylation enzymes. The alteration may also bemade by the addition of, or substitution by, one or more serine orthreonine residues to the sequence of the polypeptide (for O-linkedglycosylation sites). A variety of amino acid substitutions or deletionsat one or both of the first or third amino acid positions of aglycosylation recognition site (and/or amino acid deletion at the secondposition) results in non-glycosylation at the modified tripeptidesequence. Another means of increasing the number of carbohydratemoieties on a polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. Depending on the coupling mode used, thesugar(s) may be attached to (a) arginine and histidine; (b) freecarboxyl groups; (c) free sulfhydryl groups such as those of cysteine;(d) free hydroxyl groups such as those of serine, threonine, orhydroxyproline; (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan; or (f) the amide group of glutamine. Removal ofone or more carbohydrate moieties present on a polypeptide may beaccomplished chemically and/or enzymatically. Chemical deglycosylationmay involve, for example, exposure of a polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving the aminoacid sequence intact. Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al. [Meth. Enzymol. (1987)138:350]. The sequence of a polypeptide may be adjusted, as appropriate,depending on the type of expression system used, as mammalian, yeast,insect, and plant cells may all introduce differing glycosylationpatterns that can be affected by the amino acid sequence of the peptide.In general, heteromeric complexes of the present disclosure for use inhumans may be expressed in a mammalian cell line that provides properglycosylation, such as HEK293 or CHO cell lines, although othermammalian expression cell lines are expected to be useful as well.

The present disclosure further contemplates a method of generatingmutants, particularly sets of combinatorial mutants of an ALK4 and/or anActRIIB polypeptide as well as truncation mutants. Pools ofcombinatorial mutants are especially useful for identifying functionallyactive (e.g., TGF-beta superfamily ligand binding) ALK4 and/or ActRIIBsequences. The purpose of screening such combinatorial libraries may beto generate, for example, polypeptides variants which have alteredproperties, such as altered pharmacokinetic or altered ligand binding. Avariety of screening assays are provided below, and such assays may beused to evaluate variants. For example, ALK4:ActRIIB complex variantsmay be screened for ability to bind to one or more TGF-beta superfamilyligands to prevent binding of a TGF-beta superfamily ligand to aTGF-beta superfamily receptor, and/or to interfere with signaling causedby an TGF-beta superfamily ligand.

The activity of a ALK4:ActRIIB heteromultimer may be tested, forexample, in a cell-based or in vivo assay. For example, the effect of anALK4:ActRIIB heteromultimer on the expression of genes or activity ofproteins involved in muscle production in a muscle cell may be assessed.This may, as needed, be performed in the presence of one or moreTGF-beta superfamily ligands, and cells may be transfected so as toproduce an ALK4:ActRIIB heteromultimer, and optionally, a TGF-betasuperfamily ligand. Likewise, an ALK4:ActRIIB heteromultimer may beadministered to a mouse or other animal, and one or more measurements,such as muscle formation and strength may be assessed usingart-recognized methods. Similarly, the activity of an ALK4:ActRIIBheteromultimer, or variants thereof, may be tested, for example, inosteoblasts, adipocytes, and/or neuronal cells for any effect on growthof these cells, for example, by the assays as described herein and thoseof common knowledge in the art. A SMAD-responsive reporter gene may beused in such cell lines to monitor effects on downstream signaling.

Combinatorial-derived variants can be generated which have increasedselectivity or generally increased potency relative to a referenceALK4:ActRIIB heteromultimer. Such variants, when expressed fromrecombinant DNA constructs, can be used in gene therapy protocols.Likewise, mutagenesis can give rise to variants which have intracellularhalf-lives dramatically different than the corresponding unmodifiedALK4:ActRIIB heteromultimer. For example, the altered protein can berendered either more stable or less stable to proteolytic degradation orother cellular processes which result in destruction, or otherwiseinactivation, of an unmodified polypeptide. Such variants, and the geneswhich encode them, can be utilized to alter polypeptide complex levelsby modulating the half-life of the polypeptide. For instance, a shorthalf-life can give rise to more transient biological effects and, whenpart of an inducible expression system, can allow tighter control ofrecombinant polypeptide complex levels within the cell. In an Fc fusionprotein, mutations may be made in the linker (if any) and/or the Fcportion to alter one or more activities of the ALK4:ActRIIBheteromultimer including, for example, immunogenicity, half-life, andsolubility.

A combinatorial library may be produced by way of a degenerate libraryof genes encoding a library of polypeptides which each include at leasta portion of potential ALK4 and/or ActRIIB sequences. For instance, amixture of synthetic oligonucleotides can be enzymatically ligated intogene sequences such that the degenerate set of potential ALK4 and/orActRIIB encoding nucleotide sequences are expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display).

There are many ways by which the library of potential homologs can begenerated from a degenerate oligonucleotide sequence. Chemical synthesisof a degenerate gene sequence can be carried out in an automatic DNAsynthesizer, and the synthetic genes can then be ligated into anappropriate vector for expression. The synthesis of degenerateoligonucleotides is well known in the art [Narang, SA (1983) Tetrahedron39:3; Itakura et al. (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos.Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp 273-289; Itakuraet al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science198:1056; and Ike et al. (1983) Nucleic Acid Res. 11:477]. Suchtechniques have been employed in the directed evolution of otherproteins [Scott et al., (1990) Science 249:386-390; Roberts et al.(1992) PNAS USA 89:2429-2433; Devlin et al. (1990) Science 249: 404-406;Cwirla et al., (1990) PNAS USA 87: 6378-6382; as well as U.S. Pat. Nos.5,223,409, 5,198,346, and 5,096,815].

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, ALK4:ActRIIB heteromultimers can begenerated and isolated from a library by screening using, for example,alanine scanning mutagenesis [Ruf et al. (1994) Biochemistry33:1565-1572; Wang et al. (1994) J. Biol. Chem. 269:3095-3099; Balint etal. (1993) Gene 137:109-118; Grodberg et al. (1993) Eur. J. Biochem.218:597-601; Nagashima et al. (1993) J. Biol. Chem. 268:2888-2892;Lowman et al. (1991) Biochemistry 30:10832-10838; and Cunningham et al.(1989) Science 244:1081-1085], by linker scanning mutagenesis [Gustin etal. (1993) Virology 193:653-660; and Brown et al. (1992) Mol. Cell Biol.12:2644-2652; McKnight et al. (1982) Science 232:316], by saturationmutagenesis [Meyers et al., (1986) Science 232:613]; by PCR mutagenesis[Leung et al. (1989) Method Cell Mol Biol 1:11-19]; or by randommutagenesis, including chemical mutagenesis [Miller et al. (1992) AShort Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor,N.Y.; and Greener et al. (1994) Strategies in Mol Biol 7:32-34]. Linkerscanning mutagenesis, particularly in a combinatorial setting, is anattractive method for identifying truncated (bioactive) forms of ALK4and/or ActRIIB polypeptides.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of ALK4:ActRIIB heteromultimers. The mostwidely used techniques for screening large gene libraries typicallycomprise cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Preferredassays include TGF-beta superfamily ligand (e.g., BMP2, BMP2/7, BMP3,BMP4, BMP4/7, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, GDF3, GDF5,GDF6/BMP13, GDF7, GDF8, GDF9b/BMP15, GDF11/BMP11, GDF15/MIC1, TGF-β1,TGF-β2, TGF-β3, activin A, activin B, activin C, activin E, activin AB,activin AC, activin BC, activin AE, activin BE, nodal, glialcell-derived neurotrophic factor (GDNF), neurturin, artemin, persephin,MIS, and Lefty) binding assays and/or TGF-beta ligand-mediated cellsignaling assays.

In certain embodiments, ALK4:ActRIIB heteromultimers may furthercomprise post-translational modifications in addition to any that arenaturally present in the ALK4 and/or ActRIIB polypeptide. Suchmodifications include, but are not limited to, acetylation,carboxylation, glycosylation, phosphorylation, lipidation, andacylation. As a result, ALK4:ActRIIB heteromultimers may comprisenon-amino acid elements, such as polyethylene glycols, lipids,polysaccharide or monosaccharide, and phosphates. Effects of suchnon-amino acid elements on the functionality of a heteromultimer complexmay be tested as described herein for other heteromultimer variants.When a polypeptide of the disclosure is produced in cells by cleaving anascent form of the polypeptide, post-translational processing may alsobe important for correct folding and/or function of the protein.Different cells (e.g., CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293)have specific cellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the ALK4 and/or ActRIIB polypeptide aswell as heteromultimers comprising the same.

In certain preferred embodiments, heteromultimers described hereincomprise at least one ALK4 polypeptide associated, covalently ornon-covalently, with at least one ActRIIB polypeptide. Preferably,polypeptides disclosed herein form heterodimeric complexes, althoughhigher order heteromultimeric complexes are also included such as, butnot limited to, heterotrimers, heterotetramers, and further oligomericstructures (see, e.g., FIG. 6). In some embodiments, ALK4 and/or ActRIIBpolypeptides comprise at least one multimerization domain. As disclosedherein, the term “multimerization domain” refers to an amino acid orsequence of amino acids that promote covalent or non-covalentinteraction between at least a first polypeptide and at least a secondpolypeptide. Polypeptides disclosed herein may be joined covalently ornon-covalently to a multimerization domain. Preferably, amultimerization domain promotes interaction between a first polypeptide(e.g., an ALK4 polypeptide) and a second polypeptide (e.g., an ActRIIBpolypeptide) to promote heteromultimer formation (e.g., heterodimerformation), and optionally hinders or otherwise disfavors homomultimerformation (e.g., homodimer formation), thereby increasing the yield ofdesired heteromultimer (see, e.g., FIG. 6).

Many methods known in the art can be used to generate ALK4:ActRIIBheteromultimers. For example, non-naturally occurring disulfide bondsmay be constructed by replacing on a first polypeptide (e.g., an ALK4polypeptide) a naturally occurring amino acid with a freethiol-containing residue, such as cysteine, such that the free thiolinteracts with another free thiol-containing residue on a secondpolypeptide (e.g., an ActRIIB polypeptide) such that a disulfide bond isformed between the first and second polypeptides. Additional examples ofinteractions to promote heteromultimer formation include, but are notlimited to, ionic interactions such as described in Kjaergaard et al.,WO2007147901; electrostatic steering effects such as described in Kannanet al., U.S. Pat. No. 8,592,562; coiled-coil interactions such asdescribed in Christensen et al., U.S. 20120302737; leucine zippers suchas described in Pack & Plueckthun, (1992) Biochemistry 31: 1579-1584;and helix-turn-helix motifs such as described in Pack et al., (1993)Bio/Technology 11: 1271-1277. Linkage of the various segments may beobtained via, e.g., covalent binding such as by chemical cross-linking,peptide linkers, disulfide bridges, etc., or affinity interactions suchas by avidin-biotin or leucine zipper technology.

In certain aspects, a multimerization domain may comprise one componentof an interaction pair. In some embodiments, the polypeptides disclosedherein may form protein complexes comprising a first polypeptidecovalently or non-covalently associated with a second polypeptide,wherein the first polypeptide comprises the amino acid sequence of anALK4 polypeptide and the amino acid sequence of a first member of aninteraction pair; and the second polypeptide comprises the amino acidsequence of an ActRIIB polypeptide and the amino acid sequence of asecond member of an interaction pair. The interaction pair may be anytwo polypeptide sequences that interact to form a complex, particularlya heterodimeric complex although operative embodiments may also employan interaction pair that can form a homodimeric complex. One member ofthe interaction pair may be fused to an ALK4 or ActRIIB polypeptide asdescribed herein, including for example, a polypeptide sequencecomprising, consisting essentially of, or consisting of an amino acidsequence that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence of any one of SEQ ID NOs: 2, 3, 5, 6, 10, and 20. Aninteraction pair may be selected to confer an improved property/activitysuch as increased serum half-life, or to act as an adaptor on to whichanother moiety is attached to provide an improved property/activity. Forexample, a polyethylene glycol moiety may be attached to one or bothcomponents of an interaction pair to provide an improvedproperty/activity such as improved serum half-life.

The first and second members of the interaction pair may be anasymmetric pair, meaning that the members of the pair preferentiallyassociate with each other rather than self-associate. Accordingly, firstand second members of an asymmetric interaction pair may associate toform a heterodimeric complex (see, e.g., FIG. 6). Alternatively, theinteraction pair may be unguided, meaning that the members of the pairmay associate with each other or self-associate without substantialpreference and thus may have the same or different amino acid sequences.Accordingly, first and second members of an unguided interaction pairmay associate to form a homodimer complex or a heterodimeric complex.Optionally, the first member of the interaction pair (e.g., anasymmetric pair or an unguided interaction pair) associates covalentlywith the second member of the interaction pair. Optionally, the firstmember of the interaction pair (e.g., an asymmetric pair or an unguidedinteraction pair) associates non-covalently with the second member ofthe interaction pair.

As specific examples, the present disclosure provides fusion proteinscomprising ALK4 or ActRIIB fused to a polypeptide comprising a constantdomain of an immunoglobulin, such as a CH1, CH2, or CH3 domain of animmunoglobulin or an Fc domain. Fc domains derived from human IgG1,IgG2, IgG3, and IgG4 are provided herein. Other mutations are known thatdecrease either CDC or ADCC activity, and collectively, any of thesevariants are included in the disclosure and may be used as advantageouscomponents of a heteromultimeric complex of the disclosure. Optionally,the IgG1 Fc domain of SEQ ID NO: 31 has one or more mutations atresidues such as Asp-265, Lys-322, and Asn-434 (numbered in accordancewith the corresponding full-length IgG1). In certain cases, the mutantFc domain having one or more of these mutations (e.g., Asp-265 mutation)has reduced ability of binding to the Fcγ receptor relative to awildtype Fc domain. In other cases, the mutant Fc domain having one ormore of these mutations (e.g., Asn-434 mutation) has increased abilityof binding to the MHC class I-related Fc-receptor (FcRN) relative to awildtype Fc domain.

An example of a native amino acid sequence that may be used for the Fcportion of human IgG1 (G1Fc) is shown below (SEQ ID NO: 31). Dottedunderline indicates the hinge region, and solid underline indicatespositions with naturally occurring variants. In part, the disclosureprovides polypeptides comprising, consisting essentially of, orconsisting of amino acid sequences with 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity to SEQ ID NO: 31. Naturally occurring variants in G1Fc wouldinclude E134D and M136L according to the numbering system used in SEQ IDNO: 31 (see Uniprot P01857).

(SEQ ID NO: 31)

An example of a native amino acid sequence that may be used for the Fcportion of human IgG2 (G2Fc) is shown below (SEQ ID NO: 32). Dottedunderline indicates the hinge region and double underline indicatespositions where there are data base conflicts in the sequence (accordingto UniProt P01859). In part, the disclosure provides polypeptidescomprising, consisting essential of, or consisting of amino acidsequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 32.

(SEQ ID NO: 32)

Two examples of amino acid sequences that may be used for the Fc portionof human IgG3 (G3Fc) are shown below. The hinge region in G3Fc can be upto four times as long as in other Fc chains and contains three identical15-residue segments preceded by a similar 17-residue segment. The firstG3Fc sequence shown below (SEQ ID NO: 33) contains a short hinge regionconsisting of a single 15-residue segment, whereas the second G3Fcsequence (SEQ ID NO: 34) contains a full-length hinge region. In eachcase, dotted underline indicates the hinge region, and solid underlineindicates positions with naturally occurring variants according toUniProt P01859. In part, the disclosure provides polypeptidescomprising, consisting essential of, or consisting of amino acidsequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 33and 34.

(SEQ ID NO: 33)

(SEQ ID NO: 34)

Naturally occurring variants in G3Fc (for example, see Uniprot P01860)include E68Q, P76L, E79Q, Y81F, D97N, N100D, T124A, S169N, S169del,F221Y when converted to the numbering system used in SEQ ID NO: 33, andthe present disclosure provides fusion proteins comprising G3Fc domainscontaining one or more of these variations. In addition, the humanimmunoglobulin IgG3 gene (IGHG3) shows a structural polymorphismcharacterized by different hinge lengths [see Uniprot P01859].Specifically, variant WIS is lacking most of the V region and all of theCH1 region. It has an extra interchain disulfide bond at position 7 inaddition to the 11 normally present in the hinge region. Variant ZUClacks most of the V region, all of the CH1 region, and part of thehinge. Variant OMM may represent an allelic form or another gamma chainsubclass. The present disclosure provides additional fusion proteinscomprising G3Fc domains containing one or more of these variants.

An example of a native amino acid sequence that may be used for the Fcportion of human IgG4 (G4Fc) is shown below (SEQ ID NO: 35). Dottedunderline indicates the hinge region. In part, the disclosure providespolypeptides comprising, consisting essential of, or consisting of aminoacid sequences with 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:35.

(SEQ ID NO: 35)

A variety of engineered mutations in the Fc domain are presented hereinwith respect to the G1Fc sequence (SEQ ID NO: 31), and analogousmutations in G2Fc, G3Fc, and G4Fc can be derived from their alignmentwith G1Fc in FIG. 3. Due to unequal hinge lengths, analogous Fcpositions based on isotype alignment (FIG. 3) possess different aminoacid numbers in SEQ ID NOs: 31, 32, 33, 34, and 35. It can also beappreciated that a given amino acid position in an immunoglobulinsequence consisting of hinge, C_(H)2, and C_(H)3 regions (e.g., SEQ IDNOs: 31, 32, 33, 34, and 35) will be identified by a different numberthan the same position when numbering encompasses the entire IgG1heavy-chain constant domain (consisting of the C_(H)1, hinge, C_(H)2,and C_(H)3 regions) as in the Uniprot database. For example,correspondence between selected C_(H)3 positions in a human G1Fcsequence (SEQ ID NO: 31), the human IgG1 heavy chain constant domain(Uniprot P01857), and the human IgG1 heavy chain is as follows.

Correspondence of C_(H)3 Positions in Different Numbering Systems G1Fc(Numbering begins at first IgG1 heavy chain IgG1 heavy chain threoninein constant domain (EU numbering scheme of hinge region) (Numberingbegins at C_(H)1) Kabat et al., 1991*) Y127 Y232 Y349 S132 S237 S354E134 E239 E356 K138 K243 K360 T144 T249 T366 L146 L251 L368 N162 N267N384 K170 K275 K392 D177 D282 D399 D179 D284 D401 Y185 Y290 Y407 K187K292 K409 H213 H318 H435 K217 K322 K439 *Kabat et al. (eds) 1991; pp.688-696 in Sequences of Proteins of Immunological Interest, 5^(th) ed.,Vol. 1, NIH, Bethesda, MD.

A problem that arises in large-scale production of asymmetricimmunoglobulin-based proteins from a single cell line is known as the“chain association issue”. As confronted prominently in the productionof bispecific antibodies, the chain association issue concerns thechallenge of efficiently producing a desired multichain protein fromamong the multiple combinations that inherently result when differentheavy chains and/or light chains are produced in a single cell line[Klein et al (2012) mAbs 4:653-663]. This problem is most acute when twodifferent heavy chains and two different light chains are produced inthe same cell, in which case there are a total of 16 possible chaincombinations (although some of these are identical) when only one istypically desired. Nevertheless, the same principle accounts fordiminished yield of a desired multichain fusion protein thatincorporates only two different (asymmetric) heavy chains.

Various methods are known in the art that increase desired pairing ofFc-containing fusion polypeptide chains in a single cell line to producea preferred asymmetric fusion protein at acceptable yields [Klein et al(2012) mAbs 4:653-663; and Spiess et al (2015) Molecular Immunology67(2A): 95-106]. Methods to obtain desired pairing of Fc-containingchains include, but are not limited to, charge-based pairing(electrostatic steering), “knobs-into-holes” steric pairing, SEEDbodypairing, and leucine zipper-based pairing [Ridgway et al (1996) ProteinEng 9:617-621; Merchant et al (1998) Nat Biotech 16:677-681; Davis et al(2010) Protein Eng Des Sel 23:195-202; Gunasekaran et al (2010);285:19637-19646; Wranik et al (2012) J Biol Chem 287:43331-43339; U.S.Pat. No. 5,932,448; WO 1993/011162; WO 2009/089004, and WO 2011/034605].As described herein, these methods may be used to generateALK4-Fc:ActRIIB-Fc heteromultimer complexes. See FIG. 6.

For example, one means by which interaction between specificpolypeptides may be promoted is by engineering protuberance-into-cavity(knob-into-holes) complementary regions such as described in Arathoon etal., U.S. Pat. No. 7,183,076 and Carter et al., U.S. Pat. No. 5,731,168.“Protuberances” are constructed by replacing small amino acid sidechains from the interface of the first polypeptide (e.g., a firstinteraction pair) with larger side chains (e.g., tyrosine ortryptophan). Complementary “cavities” of identical or similar size tothe protuberances are optionally created on the interface of the secondpolypeptide (e.g., a second interaction pair) by replacing large aminoacid side chains with smaller ones (e.g., alanine or threonine). Where asuitably positioned and dimensioned protuberance or cavity exists at theinterface of either the first or second polypeptide, it is onlynecessary to engineer a corresponding cavity or protuberance,respectively, at the adjacent interface.

At neutral pH (7.0), aspartic acid and glutamic acid are negativelycharged and lysine, arginine, and histidine are positively charged.These charged residues can be used to promote heterodimer formation andat the same time hinder homodimer formation. Attractive interactionstake place between opposite charges and repulsive interactions occurbetween like charges. In part, protein complexes disclosed herein makeuse of the attractive interactions for promoting heteromultimerformation (e.g., heterodimer formation), and optionally repulsiveinteractions for hindering homodimer formation (e.g., homodimerformation) by carrying out site directed mutagenesis of chargedinterface residues.

For example, the IgG1 CH3 domain interface comprises four unique chargeresidue pairs involved in domain-domain interactions: Asp356-Lys439′,Glu357-Lys370′, Lys392-Asp399′, and Asp399-Lys409′ [residue numbering inthe second chain is indicated by (′)]. It should be noted that thenumbering scheme used here to designate residues in the IgG1 CH3 domainconforms to the EU numbering scheme of Kabat. Due to the 2-fold symmetrypresent in the CH3-CH3 domain interactions, each unique interaction willrepresented twice in the structure (e.g., Asp-399-Lys409′ andLys409-Asp399′). In the wild-type sequence, K409-D399′ favors bothheterodimer and homodimer formation. A single mutation switching thecharge polarity (e.g., K409E; positive to negative charge) in the firstchain leads to unfavorable interactions for the formation of the firstchain homodimer. The unfavorable interactions arise due to the repulsiveinteractions occurring between the same charges (negative-negative;K409E-D399′ and D399-K409E′). A similar mutation switching the chargepolarity (D399K′; negative to positive) in the second chain leads tounfavorable interactions (K409′-D399K′ and D399K-K409′) for the secondchain homodimer formation. But, at the same time, these two mutations(K409E and D399K′) lead to favorable interactions (K409E-D399K′ andD399-K409′) for the heterodimer formation.

The electrostatic steering effect on heterodimer formation and homodimerdiscouragement can be further enhanced by mutation of additional chargeresidues which may or may not be paired with an oppositely chargedresidue in the second chain including, for example, Arg355 and Lys360.The table below lists possible charge change mutations that can be used,alone or in combination, to enhance ALK4:ActRIIB heteromultimerformation.

Examples of Pair-Wise Charged Residue Mutations to Enhance HeterodimerFormation Corresponding Position in Mutation in Interacting positionmutation in second first chain first chain in second chain chain Lys409Asp or Glu Asp399′ Lys, Arg, or His Lys392 Asp or Glu Asp399′ Lys, Arg,or His Lys439 Asp or Glu Asp356′ Lys, Arg, or His Lys370 Asp or GluGlu357′ Lys, Arg, or His Asp399 Lys, Arg, or His Lys409′ Asp or GluAsp399 Lys, Arg, or His Lys392′ Asp or Glu Asp356 Lys, Arg, or HisLys439′ Asp or Glu Glu357 Lys, Arg, or His Lys370′ Asp or Glu

In some embodiments, one or more residues that make up the CH3-CH3interface in a fusion protein of the instant application are replacedwith a charged amino acid such that the interaction becomeselectrostatically unfavorable. For example, a positive-charged aminoacid in the interface (e.g., a lysine, arginine, or histidine) isreplaced with a negatively charged amino acid (e.g., aspartic acid orglutamic acid). Alternatively, or in combination with the forgoingsubstitution, a negative-charged amino acid in the interface is replacedwith a positive-charged amino acid. In certain embodiments, the aminoacid is replaced with a non-naturally occurring amino acid having thedesired charge characteristic. It should be noted that mutatingnegatively charged residues (Asp or Glu) to His will lead to increase inside chain volume, which may cause steric issues. Furthermore, Hisproton donor- and acceptor-form depends on the localized environment.These issues should be taken into consideration with the designstrategy. Because the interface residues are highly conserved in humanand mouse IgG subclasses, electrostatic steering effects disclosedherein can be applied to human and mouse IgG1, IgG2, IgG3, and IgG4.This strategy can also be extended to modifying uncharged residues tocharged residues at the CH3 domain interface.

In part, the disclosure provides desired pairing of asymmetricFc-containing polypeptide chains using Fc sequences engineered to becomplementary on the basis of charge pairing (electrostatic steering).One of a pair of Fc sequences with electrostatic complementarity can bearbitrarily fused to the ALK4 or ActRIIB polypeptide of the construct,with or without an optional linker, to generate an ALK4:ActRIIBheteromultimer. This single chain can be coexpressed in a cell of choicealong with the Fc sequence complementary to the first Fc to favorgeneration of the desired multichain construct (e.g., ALK4:ActRIIBheteromultimer). In this example based on electrostatic steering, SEQ IDNO: 23 [human G1Fc(E134K/D177K)] and SEQ ID NO: 24 [humanG1Fc(K170D/K187D)] are examples of complementary Fc sequences in whichthe engineered amino acid substitutions are double underlined, and theTGF-beta superfamily type I or type II receptor polypeptide of theconstruct can be fused to either SEQ ID NO: 23 or SEQ ID NO: 24, but notboth. Given the high degree of amino acid sequence identity betweennative hG1Fc, native hG2Fc, native hG3Fc, and native hG4Fc, it can beappreciated that amino acid substitutions at corresponding positions inhG2Fc, hG3Fc, or hG4Fc (see FIG. 3) will generate complementary Fc pairswhich may be used instead of the complementary hG1Fc pair below (SEQ IDNOs: 23 and 24).

(SEQ ID NO: 23) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101 VSNKALPAPIEKTISKAKGQ PREPQVYTLP PSRKEMTKNQ VSLTCLVKGF 151 YPSDIAVEWE SNGQPENNYKTTPPVLKSDG SFFLYSKLTV DKSRWQQGNV 201 FSCSVMHEAL HNHYTQKSLS LSPGK (SEQ IDNO: 24) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE 51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101 VSNKALPAPIEKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151 YPSDIAVEWESNGQPENNYD TTPPVLDSDG SFFLYSDLTV DKSRWQQGNV 201 FSCSVMHEAL HNHYTQKSLSLSPGK

In part, the disclosure provides desired pairing of asymmetricFc-containing polypeptide chains using Fc sequences engineered forsteric complementarity. In part, the disclosure providesknobs-into-holes pairing as an example of steric complementarity. One ofa pair of Fc sequences with steric complementarity can be arbitrarilyfused to the ALK4 or ActRIIB polypeptide of the construct, with orwithout an optional linker, to generate an ALK4:ActRIIB heteromultimer.This single chain can be co-expressed in a cell of choice along with theFc sequence complementary to the first Fc to favor generation of thedesired multi-chain construct. In this example based on knobs-into-holespairing, SEQ ID NO: 25 [human G1Fc(T144Y)] and SEQ ID NO: 26 [humanG1Fc(Y185T)] are examples of complementary Fc sequences in which theengineered amino acid substitutions are double underlined, and the ALK4or ActRIIB polypeptide of the construct can be fused to either SEQ IDNO: 25 or SEQ ID NO: 26, but not both. Given the high degree of aminoacid sequence identity between native hG1Fc, native hG2Fc, native hG3Fc,and native hG4Fc, it can be appreciated that amino acid substitutions atcorresponding positions in hG2Fc, hG3Fc, or hG4Fc (see FIG. 3) willgenerate complementary Fc pairs which may be used instead of thecomplementary hG1Fc pair below (SEQ ID NOs: 25 and 26).

(SEQ ID NO: 25) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101 VSNKALPAPIEKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLYCLVKGF 151 YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201 FSCSVMHEAL HNHYTQKSLS LSPGK (SEQ IDNO: 26) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE 51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101 VSNKALPAPIEKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151 YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLTSKLTV DKSRWQQGNV 201 FSCSVMHEAL HNHYTQKSLS LSPGK

An example of Fc complementarity based on knobs-into-holes pairingcombined with an engineered disulfide bond is disclosed in SEQ ID NO: 27[hG1Fc(S132C/T144W)] and SEQ ID NO: 28 [hG1Fc(Y127C/T144S/L146A/Y185V)].The engineered amino acid substitutions in these sequences are doubleunderlined, and the TGF-beta superfamily type I or type II polypeptideof the construct can be fused to either SEQ ID NO: 27 or SEQ ID NO: 28,but not both. Given the high degree of amino acid sequence identitybetween native hG1Fc, native hG2Fc, native hG3Fc, and native hG4Fc, itcan be appreciated that amino acid substitutions at correspondingpositions in hG2Fc, hG3Fc, or hG4Fc (see FIG. 3) will generatecomplementary Fc pairs which may be used instead of the complementaryhG1Fc pair below (SEQ ID NOs: 27 and 28).

(SEQ ID NO: 27) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101 VSNKALPAPIEKTISKAKGQ PREPQVYTLP PCREEMTKNQ VSLWCLVKGF 151 YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201 FSCSVMHEAL HNHYTQKSLS LSPGK (SEQ IDNO: 28) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE 51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101 VSNKALPAPIEKTISKAKGQ PREPQVCTLP PSREEMTKNQ VSLSCAVKGF 151 YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLVSKLTV DKSRWQQGNV 201 FSCSVMHEAL HNHYTQKSLS LSPGK

In part, the disclosure provides desired pairing of asymmetricFc-containing polypeptide chains using Fc sequences engineered togenerate interdigitating β-strand segments of human IgG and IgA C_(H)3domains. Such methods include the use of strand-exchange engineereddomain (SEED) C_(H)3 heterodimers allowing the formation of SEEDbodyfusion proteins [Davis et al. (2010) Protein Eng Design Sel 23:195-202].One of a pair of Fc sequences with SEEDbody complementarity can bearbitrarily fused to the ALK4 or ActIIB of the construct, with orwithout an optional linker, to generate an ALK4 or ActRIIB fusionpolypeptide. This single chain can be co-expressed in a cell of choicealong with the Fc sequence complementary to the first Fc to favorgeneration of the desired multi-chain construct. In this example basedon SEEDbody (Sb) pairing, SEQ ID NO: 29 [hG1Fc(Sb_(AG))] and SEQ ID NO:30 [hG1Fc(Sb_(GA))] are examples of complementary IgG Fc sequences inwhich the engineered amino acid substitutions from IgA Fc are doubleunderlined, and the ALK4 or ActRIIB polypeptide of the construct can befused to either SEQ ID NO: 29 or SEQ ID NO: 30, but not both. Given thehigh degree of amino acid sequence identity between native hG1Fc, nativehG2Fc, native hG3Fc, and native hG4Fc, it can be appreciated that aminoacid substitutions at corresponding positions in hG1Fc, hG2Fc, hG3Fc, orhG4Fc (see FIG. 3) will generate an Fc monomer which may be used in thecomplementary IgG-IgA pair below (SEQ ID NOs: 29 and 30).

(SEQ ID NO: 29) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101 VSNKALPAPIEKTISKAKGQ PFRPEVHLLP PSREEMTKNQ VSLTCLARGF 151 YPKDIAVEWE SNGQPENNYKTTPSRQEPSQ GTTTFAVTSK LTVDKSRWQQ 201 GNVFSCSVMH EALHNHYTQK TISLSPGK (SEQID NO: 30) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE 51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101 VSNKALPAPIEKTISKAKGQ PREPQVYTLP PPSEELALNE LVTLTCLVKG 151 FYPSDIAVEWESNGQELPRE KYLTWAPVLD SDGSFFLYSI LRVAAEDWKK 201 GDTFSCSVMH EALHNHYTQKSLDRSPGK

In part, the disclosure provides desired pairing of asymmetricFc-containing polypeptide chains with a cleavable leucine zipper domainattached at the C-terminus of the Fc C_(H)3 domains. Attachment of aleucine zipper is sufficient to cause preferential assembly ofheterodimeric antibody heavy chains [Wranik et al (2012) J Biol Chem287:43331-43339]. As disclosed herein, one of a pair of Fc sequencesattached to a leucine zipper-forming strand can be arbitrarily fused tothe ALK4 or ActRIIB polypeptide of the construct, with or without anoptional linker, to generate an ALK4 or ActRIIB fusion polypeptide. Thissingle chain can be co-expressed in a cell of choice along with the Fcsequence attached to a complementary leucine zipper-forming strand tofavor generation of the desired multi-chain construct. Proteolyticdigestion of the construct with the bacterial endoproteinase Lys-C postpurification can release the leucine zipper domain, resulting in an Fcconstruct whose structure is identical to that of native Fc. In thisexample based on leucine zipper pairing, SEQ ID NO: 36 [hG1Fc-Ap1(acidic)] and SEQ ID NO: 37 [hG1Fc-Bp1 (basic)] are examples ofcomplementary IgG Fc sequences in which the engineered complimentaryleucine zipper sequences are underlined, and the ALK4 or ActRIIBpolypeptide of the construct can be fused to either SEQ ID NO: 36 or SEQID NO: 37, but not both. Given the high degree of amino acid sequenceidentity between native hG1Fc, native hG2Fc, native hG3Fc, and nativehG4Fc, it can be appreciated that leucine zipper-forming sequencesattached, with or without an optional linker, to hG1Fc, hG2Fc, hG3Fc, orhG4Fc (see FIG. 3) will generate an Fc monomer which may be used in thecomplementary leucine zipper-forming pair below (SEQ ID NOs: 36 and 37).

(SEQ ID NO: 36) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101 VSNKALPAPIEKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151 YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201 FSCSVMHEAL HNHYTQKSLSLSPGKGGSAQ LEKELQALEKP ENAQLEWELQ 251 ALEKELAQGA T (SEQ ID NO: 37) 1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE 51 VKFNWYVDGVEVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101 VSNKALPAPI EKTISKAKGQPREPQVYTLP PSREEMTKNQ VSLTCLVKGF 151 YPSDIAVEWE SNGQPENNYK TTPPVLDSDGSFFLYSKLTV DKSRWQQGNV 201 FSCSVMHEAL HNHYTQKSLSLSPGKGGSAQ LKKKLQALKK KNAQLKWKLQ 251 ALKKKLAQGA T

In certain aspects, the disclosure relates to ALK4 polypeptides (e.g.,ALK4-Fc fusion proteins) comprising one or more amino acid modificationsthat alter the isoelectric point (pI) of the ALK4 polypeptide and/orActRIIB polypeptides (e.g., ActRIIB-Fc fusion proteins) comprising oneor more amino acid modifications that alter the isoelectric point of theActRIIB polypeptide. In some embodiments, one or more candidate domainsthat have a pI value higher than about 5.0, 5.5, 6.0, 6.5, 7.0, 7.5,8.0, 8.5, or 9.0 are selected for construction of the full multidomainprotein. In other embodiments, one or more candidate domains that have apI value less than about 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, or 5.0are selected for construction of the full multidomain protein. It willbe understood by one skilled in the art that a single protein will havemultiple charge forms. Without wishing to be bound by any particulartheory, the charge of a protein can be modified by a number of differentmechanisms including but not limited to, amino acid substitution,cationization, deamination, carboxyl-terminal amino acid heterogeneity,phosphorylation and glycosylation.

The pI of a protein may be determined by a variety of methods includingbut not limited to, isoelectric focusing and various computer algorithms(see for example Bjellqvist et al., 1993, Electrophoresis 14:1023). Inone embodiment, pI is determined using a Pharmacia Biotech Multiphor 2electrophoresis system with a multi temp refrigerated bath recirculationunit and an EPS 3501 XL power supply. Pre-cast ampholine gels (e.g.,Amersham Biosciences, pI range 2.5-10) are loaded with protein samples.Broad range pI marker standards (e.g., Amersham, pI range 3-10, 8 .mu.L)are used to determine relative pI for the proteins. Electrophoresis isperformed, for example, at 1500 V, 50 mA for 105 minutes. The gel isfixed using, for example, a Sigma fixing solution (5×) diluted withpurified water to 1× Staining is performed, for example, overnight atroom temperature using Simply Blue stain (Invitrogen). Destaining iscarried out, for example, with a solution that consisted of 25% ethanol,8% acetic acid and 67% purified water. Isoelectric points are determinedusing, for example, a Bio-Rad Densitometer relative to calibrationcurves of the standards. The one or more metrics may further includemetrics characterizing stability of the domain under one or moredifferent conditions selected from the group consisting of different pHvalues, different temperatures, different shear stresses, and differentfreeze/thaw cycles.

In part, the disclosure provides desired pairing of asymmetricFc-containing polypeptide chains by methods described above incombination with additional mutations in the Fc domain which facilitatepurification of the desired heteromeric species. An example iscomplementarity of Fc domains based on knobs-into-holes pairing combinedwith an engineered disulfide bond, as disclosed in SEQ ID NOs: 27-28,plus additional substitution of two negatively charged amino acids(aspartic acid or glutamic acid) in one Fc-containing polypeptide chainand two positively charged amino acids (e.g., arginine) in thecomplementary Fc-containing polypeptide chain (SEQ ID NOs: 66-67). Thesefour amino acid substitutions facilitate selective purification of thedesired heteromeric fusion protein from a heterogeneous polypeptidemixture based on differences in isoelectric point or net molecularcharge. The engineered amino acid substitutions in these sequences aredouble underlined below, and the ALK4 or ActRIIB polypeptide of theconstruct can be fused to either SEQ ID NO: 66 or SEQ ID NO: 67, but notboth. Given the high degree of amino acid sequence identity betweennative hG1Fc, native hG2Fc, native hG3Fc, and native hG4Fc, it can beappreciated that amino acid substitutions at corresponding positions inhG2Fc, hG3Fc, or hG4Fc (see FIG. 3) will generate complementary Fc pairswhich may be used instead of the complementary hG1Fc pair below (SEQ IDNOs: 66-67).

(SEQ ID NO: 66) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101 VSNKALPAPIEKTISKAKGQ PREPQVYTLP PCREEMTENQ VSLWCLVKGF 151 YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201 FSCSVMHEAL HNHYTQDSLS LSPGK (SEQ IDNO: 67) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE 51VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101 VSNKALPAPIEKTISKAKGQ PREPQVCTLP PSREEMTKNQ VSLSCAVKGF 151 YPSDIAVEWE SRGQPENNYKTTPPVLDSRG SFFLVSKLTV DKSRWQQGNV 201 FSCSVMHEAL HNHYTQKSLS LSPGK

Another example involves complementarity of Fc domains based onknobs-into-holes pairing combined with an engineered disulfide bond, asdisclosed in SEQ ID NOs: 27-28, plus a histidine-to-argininesubstitution at position 213 in one Fc-containing polypeptide chain (SEQID NO: 68). This substitution (denoted H435R in the numbering system ofKabat et al.) facilitates separation of desired heteromer fromundesirable homodimer based on differences in affinity for protein A.The engineered amino acid substitution is indicated by double underline,and the ALK4 or ActRIIB polypeptide of the construct can be fused toeither SEQ ID NO: 68 or SEQ ID NO: 28, but not both. Given the highdegree of amino acid sequence identity between native hG1Fc, nativehG2Fc, native hG3Fc, and native hG4Fc, it can be appreciated that aminoacid substitutions at corresponding positions in hG2Fc, hG3Fc, or hG4Fc(see FIG. 3) will generate complementary Fc pairs which may be usedinstead of the complementary hG1Fc pair of SEQ ID NO: 68 (below) and SEQID NO: 28.

(SEQ ID NO: 68) 1 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE51 VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK 101 VSNKALPAPIEKTISKAKGQ PREPQVYTLP PCREEMTKNQ VSLWCLVKGF 151 YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSKLTV DKSRWQQGNV 201 FSCSVMHEAL HNRYTQKSLS LSPGK

A variety of engineered mutations in the Fc domain are presented abovewith respect to the G1Fc sequence (SEQ ID NO: 31). Analogous mutationsin G2Fc, G3Fc, and G4Fc can be derived from their alignment with G1Fc inFIG. 3. Due to unequal hinge lengths, analogous Fc positions based onisotype alignment (FIG. 3) possess different amino acid numbers in SEQID NOs: 31, 32, 33, 34, and 35 as summarized in the following table.

Correspondence between C_(H)3 Positions for Human Fc Isotypes* IgG1 SEQID IgG4 IgG2 IgG3 NO: 31 SEQ ID NO: 35 SEQ ID NO: 32 SEQ ID NO: 33Numbering Numbering Numbering Numbering begins at begins at begins atbegins at THT . . . ESK . . . VEC . . . EPK . . . Y127 Y131 Y125 Y134S132 S136 S130 S139 E134 E138 E132 E141 K138 K142 K136 K145 T144 T148T142 T151 L146 L150 L144 L153 N162 N166 N160 S169 K170 K174 K168 N177D177 D181 D175 D184 D179 D183 D177 D186 Y185 Y189 Y183 Y192 K187 R191K185 K194 H213 H217 H211 R220 K217 K221 K215 K224 *Numbering based onmultiple sequence alignment shown in FIG. 3

It is understood that different elements of the fusion proteins (e.g.,immunoglobulin Fc fusion proteins) may be arranged in any manner that isconsistent with desired functionality. For example, an ALK4 and/orActRIIB polypeptide domain may be placed C-terminal to a heterologousdomain, or alternatively, a heterologous domain may be placed C-terminalto an ALK4 and/or ActRIIB polypeptide domain. The ALK4 and/or ActRIIBpolypeptide domain and the heterologous domain need not be adjacent in afusion protein, and additional domains or amino acid sequences may beincluded C- or N-terminal to either domain or between the domains.

For example, an ALK4 and/or ActRIIB receptor fusion protein may comprisean amino acid sequence as set forth in the formula A-B-C. The B portioncorresponds to an ALK4 or ActRIIB polypeptide domain. The A and Cportions may be independently zero, one, or more than one amino acid,and both the A and C portions when present are heterologous to B. The Aand/or C portions may be attached to the B portion via a linkersequence. A linker may be rich in glycine (e.g., 2-10, 2-5, 2-4, 2-3glycine residues) or glycine and proline residues and may, for example,contain a single sequence of threonine/serine and glycines or repeatingsequences of threonine/serine and/or glycines, e.g., GGG (SEQ ID NO:13), GGGG (SEQ ID NO: 14), TGGGG (SEQ ID NO: 15), SGGGG (SEQ ID NO: 16),TGGG (SEQ ID NO: 17), SGGG (SEQ ID NO: 18), or GGGGS (SEQ ID NO: 58)singlets, or repeats. In certain embodiments, an ALK4 and/or ActRIIBfusion protein comprises an amino acid sequence as set forth in theformula A-B-C, wherein A is a leader (signal) sequence, B consists of anALK4 and/or ActRIIB polypeptide domain, and C is a polypeptide portionthat enhances one or more of in vivo stability, in vivo half-life,uptake/administration, tissue localization or distribution, formation ofprotein complexes, and/or purification. In certain embodiments, an ALK4and/or ActRIIB fusion protein comprises an amino acid sequence as setforth in the formula A-B-C, wherein A is a TPA leader sequence, Bconsists of a ALK4 or ActRIIB receptor polypeptide domain, and C is animmunoglobulin Fc domain. Preferred fusion proteins comprise the aminoacid sequence set forth in any one of SEQ ID NOs: 39, 41, 42, 44, 45,46, 47, 48, 70, 72, 74, 76, 78, and 80.

In some embodiments, ALK4:ActRIIB heteromultimers further comprise oneor more heterologous portions (domains) so as to confer a desiredproperty. For example, some fusion domains are particularly useful forisolation of the fusion proteins by affinity chromatography. Well-knownexamples of such fusion domains include, but are not limited to,polyhistidine, Glu-Glu, glutathione S-transferase (GST), thioredoxin,protein A, protein G, an immunoglobulin heavy-chain constant region(Fc), maltose binding protein (MBP), or human serum albumin. For thepurpose of affinity purification, relevant matrices for affinitychromatography, such as glutathione-, amylase-, and nickel- orcobalt-conjugated resins are used. Many of such matrices are availablein “kit” form, such as the Pharmacia GST purification system and theQIAexpress system (Qiagen) useful with (HIS₆ (SEQ ID NO: 84)) fusionpartners. As another example, a fusion domain may be selected so as tofacilitate detection of the ligand trap polypeptides. Examples of suchdetection domains include the various fluorescent proteins (e.g., GFP)as well as “epitope tags,” which are usually short peptide sequences forwhich a specific antibody is available. Well-known epitope tags forwhich specific monoclonal antibodies are readily available include FLAG,influenza virus haemagglutinin (HA), and c-myc tags. In some cases, thefusion domains have a protease cleavage site, such as for factor Xa orthrombin, which allows the relevant protease to partially digest thefusion proteins and thereby liberate the recombinant proteins therefrom.The liberated proteins can then be isolated from the fusion domain bysubsequent chromatographic separation.

In certain embodiments, ALK4 and/or ActRIIB polypeptides may contain oneor more modifications that are capable of stabilizing the polypeptides.For example, such modifications enhance the in vitro half-life of thepolypeptides, enhance circulatory half-life of the polypeptides, and/orreduce proteolytic degradation of the polypeptides. Such stabilizingmodifications include, but are not limited to, fusion proteins(including, for example, fusion proteins comprising an ALK4 and/orActRIIB polypeptide domain and a stabilizer domain), modifications of aglycosylation site (including, for example, addition of a glycosylationsite to a polypeptide of the disclosure), and modifications ofcarbohydrate moiety (including, for example, removal of carbohydratemoieties from a polypeptide of the disclosure). As used herein, the term“stabilizer domain” not only refers to a fusion domain (e.g., animmunoglobulin Fc domain) as in the case of fusion proteins, but alsoincludes nonproteinaceous modifications such as a carbohydrate moiety,or nonproteinaceous moiety, such as polyethylene glycol.

In preferred embodiments, ALK4:ActRIIB heteromultimers to be used inaccordance with the methods described herein are isolated complexes. Asused herein, an isolated protein (or protein complex) or polypeptide (orpolypeptide complex) is one which has been separated from a component ofits natural environment. In some embodiments, a heteromultimer of thedisclosure is purified to greater than 95%, 96%, 97%, 98%, or 99% purityas determined by, for example, electrophoretic (e.g., SDS-PAGE,isoelectric focusing (IEF), capillary electrophoresis) orchromatographic (e.g., ion exchange or reverse phase HPLC). Methods forassessment of antibody purity are well known in the art [Flatman et al.,(2007) J. Chromatogr. B 848:79-87]. In some embodiments, ALK4:ActRIIBheteromultimer preparations are substantially free of ALK4 and/orActRIIB homomultimers. For example, in some embodiments, ALK4:ActRIIBheteromultimer preparations comprise less than about 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, or less than 1% ALK4 homomultimers. In someembodiments, ALK4:ActRIIB heteromultimer preparations comprise less thanabout 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% ActRIIBhomomultimers. In some embodiments, ALK4:ActRIIB heteromultimerpreparations comprise less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, or less than 1% ALK4 homomultimers and less than about 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, or less than 1% ActRIIB homomultimers.

In certain embodiments, ALK4 and/or ActRIIB polypeptides, as well asheteromultimers comprising the same, of the disclosure can be producedby a variety of art-known techniques. For example, polypeptides can besynthesized using standard protein chemistry techniques such as thosedescribed in Bodansky, M. Principles of Peptide Synthesis, SpringerVerlag, Berlin (1993) and Grant G. A. (ed.), Synthetic Peptides: AUser's Guide, W. H. Freeman and Company, New York (1992). In addition,automated peptide synthesizers are commercially available (AdvancedChemTech Model 396; Milligen/Biosearch 9600). Alternatively, thepolypeptides, including fragments or variants thereof, may berecombinantly produced using various expression systems [E. coli,Chinese Hamster Ovary (CHO) cells, COS cells, baculovirus] as is wellknown in the art. In a further embodiment, the modified or unmodifiedpolypeptides may be produced by digestion of recombinantly producedfull-length ALK4 and/or ActRIIB polypeptides by using, for example, aprotease, e.g., trypsin, thermolysin, chymotrypsin, pepsin, or pairedbasic amino acid converting enzyme (PACE). Computer analysis (usingcommercially available software, e.g., MacVector, Omega, PCGene,Molecular Simulation, Inc.) can be used to identify proteolytic cleavagesites.

3. Nucleic Acids Encoding ALK4 and/or ActRIIB Polypeptides

In certain embodiments, the present disclosure provides isolated and/orrecombinant nucleic acids encoding ALK4 and/or ActRIIB polypeptides(including fragments, functional variants, and fusion proteins thereof)disclosed herein. For example, SEQ ID NO: 11 encodes a naturallyoccurring human ALK4 precursor polypeptide, while SEQ ID NO: 12 encodesa processed extracellular domain of ALK4. The subject nucleic acids maybe single-stranded or double stranded. Such nucleic acids may be DNA orRNA molecules. These nucleic acids may be used, for example, in methodsfor making ALK4:ActRIIB heteromultimers as described herein.

As used herein, isolated nucleic acid(s) refers to a nucleic acidmolecule that has been separated from a component of its naturalenvironment. An isolated nucleic acid includes a nucleic acid moleculecontained in cells that ordinarily contain the nucleic acid molecule,but the nucleic acid molecule is present extrachromosomally or at achromosomal location that is different from its natural chromosomallocation.

In certain embodiments, nucleic acids encoding ALK4 and/or ActRIIBpolypeptides of the present disclosure are understood to include any oneof SEQ ID NOs: 7, 8, 11, 12, 21, 22, 40, 43, 71, 73, 75, 77, 79, 81, 82,or 83, as well as variants thereof. Variant nucleotide sequences includesequences that differ by one or more nucleotide substitutions,additions, or deletions including allelic variants, and therefore, willinclude coding sequences that differ from the nucleotide sequencedesignated in any one of SEQ ID NOs: 7, 8, 11, 12, 21, 22, 40, 43, 71,73, 75, 77, 79, 81, 82, or 83.

In certain embodiments, ALK4 and/or ActRIIB polypeptides of the presentdisclosure are encoded by isolated or recombinant nucleic acid sequencesthat comprise, consist essentially of, or consists of a sequence that isleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 7, 8, 11, 12,21, 22, 40, 43, 71, 73, 75, 77, 79, 81, 82, or 83. One of ordinary skillin the art will appreciate that nucleic acid sequences that comprise,consist essentially of, or consists of a sequence complementary to asequence that is least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs:7, 8, 11, 12, 21, 22, 40, 43, 71, 73, 75, 77, 79, 81, 82, or 83 alsowithin the scope of the present disclosure. In further embodiments, thenucleic acid sequences of the disclosure can be isolated, recombinant,and/or fused with a heterologous nucleotide sequence or in a DNAlibrary.

In other embodiments, nucleic acids of the present disclosure alsoinclude nucleotide sequences that hybridize under stringent conditionsto the nucleotide sequence designated in SEQ ID NOs: 7, 8, 11, 12, 21,22, 40, 43, 71, 73, 75, 77, 79, 81, 82, or 83, the complement sequenceof SEQ ID NOs: 7, 8, 11, 12, 21, 22, 40, 43, 71, 73, 75, 77, 79, 81, 82,or 83, or fragments thereof. One of ordinary skill in the art willunderstand readily that appropriate stringency conditions which promoteDNA hybridization can be varied. For example, one could perform thehybridization at 6.0×sodium chloride/sodium citrate (SSC) at about 45°C., followed by a wash of 2.0×SSC at 50° C. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or temperature or salt concentration may be held constant whilethe other variable is changed. In one embodiment, the disclosureprovides nucleic acids which hybridize under low stringency conditionsof 6×SSC at room temperature followed by a wash at 2×SSC at roomtemperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NOs: 7, 8, 11, 12, 21, 22, 40, 43, 71, 73, 75, 77, 79, 81, 82,or 83 to degeneracy in the genetic code are also within the scope of thedisclosure. For example, a number of amino acids are designated by morethan one triplet. Codons that specify the same amino acid, or synonyms(for example, CAU and CAC are synonyms for histidine) may result in“silent” mutations which do not affect the amino acid sequence of theprotein. However, it is expected that DNA sequence polymorphisms that dolead to changes in the amino acid sequences of the subject proteins willexist among mammalian cells. One skilled in the art will appreciate thatthese variations in one or more nucleotides (up to about 3-5% of thenucleotides) of the nucleic acids encoding a particular protein mayexist among individuals of a given species due to natural allelicvariation. Any and all such nucleotide variations and resulting aminoacid polymorphisms are within the scope of this disclosure.

In certain embodiments, the recombinant nucleic acids of the disclosuremay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the disclosure. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In some embodiments, the expression vector contains aselectable marker gene to allow the selection of transformed host cells.Selectable marker genes are well known in the art and will vary with thehost cell used.

In certain aspects, the subject nucleic acid is provided in anexpression vector comprising a nucleotide sequence encoding an ALK4and/or ActRIIB polypeptide and operably linked to at least oneregulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of ALK4 and/or ActRIIB polypeptide.Accordingly, the term regulatory sequence includes promoters, enhancers,and other expression control elements. Exemplary regulatory sequencesare described in Goeddel; Gene Expression Technology: Methods inEnzymology, Academic Press, San Diego, Calif. (1990). For instance, anyof a wide variety of expression control sequences that control theexpression of a DNA sequence when operatively linked to it may be usedin these vectors to express DNA sequences encoding a ALK4 and/or ActRIIBpolypeptides. Such useful expression control sequences, include, forexample, the early and late promoters of SV40, tet promoter, adenovirusor cytomegalovirus immediate early promoter, RSV promoters, the lacsystem, the trp system, the TAC or TRC system, T7 promoter whoseexpression is directed by T7 RNA polymerase, the major operator andpromoter regions of phage lambda, the control regions for fd coatprotein, the promoter for 3-phosphoglycerate kinase or other glycolyticenzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters ofthe yeast α-mating factors, the polyhedron promoter of the baculovirussystem and other sequences known to control the expression of genes ofprokaryotic or eukaryotic cells or their viruses, and variouscombinations thereof. It should be understood that the design of theexpression vector may depend on such factors as the choice of the hostcell to be transformed and/or the type of protein desired to beexpressed. Moreover, the vector's copy number, the ability to controlthat copy number and the expression of any other protein encoded by thevector, such as antibiotic markers, should also be considered.

A recombinant nucleic acid of the present disclosure can be produced byligating the cloned gene, or a portion thereof, into a vector suitablefor expression in either prokaryotic cells, eukaryotic cells (yeast,avian, insect or mammalian), or both. Expression vehicles for productionof a recombinant ALK4 and/or ActRIIB polypeptides include plasmids andother vectors. For instance, suitable vectors include plasmids of thefollowing types: pBR322-derived plasmids, pEMBL-derived plasmids,pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmidsfor expression in prokaryotic cells, such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures [Molecular Cloning A Laboratory Manual, 3rd Ed.,ed. by Sambrook, Fritsch and Maniatis Cold Spring Harbor LaboratoryPress, 2001]. In some instances, it may be desirable to express therecombinant polypeptides by the use of a baculovirus expression system.Examples of such baculovirus expression systems include pVL-derivedvectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors(such as pAcUW1), and pBlueBac-derived vectors (such as the ß-galcontaining pBlueBac III).

In a preferred embodiment, a vector will be designed for production ofthe subject ALK4 and/or ActRIIB polypeptides in CHO cells, such as aPcmv-Script vector (Stratagene, La Jolla, Calif.), pcDNA4 vectors(Invitrogen, Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison,Wis.). As will be apparent, the subject gene constructs can be used tocause expression of the subject ALK4 and/or ActRIIB polypeptide in cellspropagated in culture, e.g., to produce proteins, including fusionproteins or variant proteins, for purification.

This disclosure also pertains to a host cell transfected with arecombinant gene including a coding sequence for one or more of thesubject ALK4 and/or ActRIIB polypeptides. The host cell may be anyprokaryotic or eukaryotic cell. For example, an ALK4 and/or ActRIIBpolypeptide may be expressed in bacterial cells such as E. coli, insectcells (e.g., using a baculovirus expression system), yeast, or mammaliancells [e.g. a Chinese hamster ovary (CHO) cell line]. Other suitablehost cells are known to those skilled in the art.

Accordingly, the present disclosure further pertains to methods ofproducing the subject ALK4 and/or ActRIIB polypeptides. For example, ahost cell transfected with an expression vector encoding an ALK4 and/orActRIIB polypeptide can be cultured under appropriate conditions toallow expression of the ALK4 and/or ActRIIB polypeptide to occur. Thepolypeptide may be secreted and isolated from a mixture of cells andmedium containing the polypeptide. Alternatively, ALK4 and/or ActRIIBpolypeptide may be isolated from a cytoplasmic or membrane fractionobtained from harvested and lysed cells. A cell culture includes hostcells, media and other byproducts. Suitable media for cell culture arewell known in the art. The subject polypeptides can be isolated fromcell culture medium, host cells, or both, using techniques known in theart for purifying proteins, including ion-exchange chromatography, gelfiltration chromatography, ultrafiltration, electrophoresis,immunoaffinity purification with antibodies specific for particularepitopes of ALK4 and/or ActRIIB polypeptides and affinity purificationwith an agent that binds to a domain fused to ALK4 and/or ActRIIBpolypeptide (e.g., a protein A column may be used to purify ALK4-Fcand/or ActRIIB-Fc fusion proteins). In some embodiments, the ALK4 and/orActRIIB polypeptide is a fusion protein containing a domain whichfacilitates its purification.

In some embodiments, purification is achieved by a series of columnchromatography steps, including, for example, three or more of thefollowing, in any order: protein A chromatography, Q sepharosechromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange. An ALK4and/or ActRIIB polypeptides, as well as fusion proteins thereof, may bepurified to a purity of >90%, >95%, >96%, >98%, or >99% as determined bysize exclusion chromatography and >90%, >95%, >96%, >98%, or >99% asdetermined by SDS PAGE. The target level of purity should be one that issufficient to achieve desirable results in mammalian systems,particularly non-human primates, rodents (mice), and humans.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant ALK4 and/orActRIIB polypeptide, can allow purification of the expressed fusionprotein by affinity chromatography using a Ni²⁺ metal resin. Thepurification leader sequence can then be subsequently removed bytreatment with enterokinase to provide the purified ALK4 and/or ActRIIBpolypeptide, as well as heteromultimers thereof [Hochuli et al. (1987)J. Chromatography 411:177; and Janknecht et al. (1991) PNAS USA88:8972].

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence. See,e.g., Current Protocols in Molecular Biology, eds. Ausubel et al., JohnWiley & Sons: 1992.

4. Exemplary Therapeutic Uses

In certain embodiments, an ALK4:ActRIIB heteromultimer (e.g.,ALK4:ActRIIB heterodimers) can be used to treat or prevent a disease orcondition that is associated with abnormal activity of anALK4:ActRIIB-binding ligand. These diseases, disorders, or conditionsare generally referred to herein as “ALK4:ActRIIB-associated conditions”or “ALK4:ActRIIB-associated disorders.” In certain embodiments, thepresent disclosure provides methods of treating or preventing anALK4:ActRIIB-associated condition in an individual by administering toan individual in need thereof a therapeutically effective amount of anALK4:ActRIIB heteromultimer. The terms “subject,” an “individual,” or a“patient” are interchangeable throughout the specification. Any of theALK4:ActRIIB heteromultimers of the disclosure can potentially beemployed individually or in combination for therapeutic uses disclosedherein. These methods are particularly aimed at therapeutic andprophylactic treatments of mammals including, for example, rodents,primates, and humans.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. The term “treating” as used hereinincludes amelioration or elimination of the condition once it has beenestablished. In either case, prevention or treatment may be discerned inthe diagnosis provided by a physician or other health care provider andthe intended result of administration of the therapeutic agent.

In general, treatment or prevention of a disease or condition asdescribed in the present disclosure is achieved by administering anALK4:ActRIIB heteromultimer of the present disclosure in an “effectiveamount”. An effective amount of an agent refers to an amount effective,at dosages and for periods of time necessary, to achieve the desiredtherapeutic or prophylactic result. A “therapeutically effective amount”of an agent of the present disclosure may vary according to factors suchas the disease state, age, sex, and weight of the individual, and theability of the agent to elicit a desired response in the individual. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result.

Naturally occurring ALK4 and ActRIIB receptor-ligand complexes playessential roles in tissue growth as well as early developmentalprocesses such as the correct formation of various structures or in oneor more post-developmental capacities including sexual development,pituitary hormone production, and creation of bone and cartilage. Thus,ALK4:ActRIIB-associated conditions include, but are not limited to,abnormal tissue growth and developmental defects. In addition,ALK4:ActRIIB-associated conditions include, but are not limited to,disorders of cell growth and differentiation such as inflammation,allergy, autoimmune diseases, and tumors.

For example, ALK4:ActRIIB-associated conditions include neuromusculardisorders (e.g., muscular dystrophy and muscle atrophy), congestiveobstructive pulmonary disease (and muscle wasting associated with COPD),muscle wasting syndrome, sarcopenia, cachexia, adipose tissue disorders(e.g., obesity), type 2 diabetes (NIDDM, adult-onset diabetes), and bonedegenerative disease (e.g., osteoporosis). Other exemplaryALK4:ActRIIB-associated conditions include musculodegenerative andneuromuscular disorders, tissue repair (e.g., wound healing),neurodegenerative diseases (e.g., amyotrophic lateral sclerosis), andimmunologic disorders (e.g., disorders related to abnormal proliferationor function of lymphocytes).

In some embodiments, the ALK4:ActRIIB-associated condition is aninterstitial lung disease (e.g., idiopathic pulmonary fibrosis). In someembodiments, the interstitial lung disease is pulmonary fibrosis. Insome embodiments, the interstitial lung disease is caused by any one ofthe following: silicosis, asbestosis, berylliosis, hypersensitivitypneumonitis, drug use (e.g., antibiotics, chemotherapeutic drugs,antiarrhythmic agents, statins), systemic sclerosis, polymyositis,dermatomyositis, systemic lupus erythematosus, rheumatoid arthritis, aninfection (e.g., atypical pneumonia, pneumocystis pneumonia,tuberculosis, Chlamydia trachomatis, and/or respiratory syncytialvirus), lymphangitic carcinomatosis, cigarette smoking, or developmentaldisorders. In some embodiments, the interstitial lung disease isidiopathic (e.g., sarcoidosis, idiopathic pulmonary fibrosis,Hamman-Rich syndrome, and/or antisynthetase syndrome). In particularembodiments, the interstitial lung disease is idiopathic pulmonaryfibrosis. In some embodiments, the treatment for idiopathic pulmonaryfibrosis is administered in combination with an additional therapeuticagent. In some embodiments, the additional therapeutic agent is selectedfrom the group consisting of: pirfenidone, N-acetylcysteine, prednisone,azathioprine, nintedanib, derivatives thereof and combinations thereof.

In certain embodiments, an ALK4:ActRIIB heteromultimer of the disclosuremay be used as part of a treatment for a muscular dystrophy. The term“muscular dystrophy” refers to a group of degenerative muscle diseasescharacterized by gradual weakening and deterioration of skeletal musclesand sometimes the heart and respiratory muscles. Muscular dystrophiesare genetic disorders characterized by progressive muscle wasting andweakness that begin with microscopic changes in the muscle. As musclesdegenerate over time, the person's muscle strength declines. Exemplarymuscular dystrophies that can be treated with a regimen including thesubject TGF-beta superfamily heteromultimer complexes include: Duchennemuscular dystrophy (DMD), Becker muscular dystrophy (BMD),Emery-Dreifuss muscular dystrophy (EDMD), limb-girdle muscular dystrophy(LGMD), facioscapulohumeral muscular dystrophy (FSH or FSHD) (also knownas Landouzy-Dejerine), myotonic dystrophy (MMD; also known as Steinert'sDisease), oculopharyngeal muscular dystrophy (OPMD), distal musculardystrophy (DD), congenital muscular dystrophy (CMD).

Duchenne muscular dystrophy (DMD) was first described by the Frenchneurologist Guillaume Benjamin Amand Duchenne in the 1860s. Beckermuscular dystrophy (BMD) is named after the German doctor Peter EmilBecker, who first described this variant of DMD in the 1950s. DMD is oneof the most frequent inherited diseases in males, affecting one in 3,500boys. DMD occurs when the dystrophin gene, located on the short arm ofthe X chromosome, is defective. Since males only carry one copy of the Xchromosome, they only have one copy of the dystrophin gene. Without thedystrophin protein, muscle is easily damaged during cycles ofcontraction and relaxation. While early in the disease musclecompensates by regeneration, later on muscle progenitor cells cannotkeep up with the ongoing damage and healthy muscle is replaced bynon-functional fibro-fatty tissue.

BMD results from different mutations in the dystrophin gene. BMDpatients have some dystrophin, but it is either of insufficient quantityor poor quality. The presence of some dystrophin protects the muscles ofpatients with BMD from degenerating as severely or as quickly as thoseof patients with DMD.

Studies in animals indicate that inhibition of the GDF8 signalingpathway may effectively treat various aspects of disease in DMD and BMDpatients (Bogdanovich et al., 2002, Nature 420:418-421; Pistilli et al.,2011, Am J Pathol 178:1287-1297). Thus, ALK4:ActRIIB antagonists of thedisclosure may act as GDF8 inhibitors (antagonists), and constitute analternative means of blocking signaling by GDF8 and/or related TGFβsuperfamily ligands in vivo in DMD and BMD patients.

Similarly, ALK4:ActRIIB heteromultimers of the disclosure may provide aneffective means to increase muscle mass in other disease conditions thatare in need of muscle growth. For example, amyotrophic lateral sclerosis(ALS), also called Lou Gehrig's disease or motor neuron disease, is achronic, progressive, and incurable CNS disorder that attacks motorneurons, which are components of the central nervous system required forinitiation of skeletal muscle contraction. In ALS, motor neuronsdeteriorate and eventually die, and though a person's brain normallyremains fully functioning and alert, initiation of muscle contraction isblocked at the spinal level. Individuals who develop ALS are typicallybetween 40 and 70 years old, and the first motor neurons to degenerateare those innervating the arms or legs. Patients with ALS may havetrouble walking, may drop things, fall, slur their speech, and laugh orcry uncontrollably. As the disease progresses, muscles in the limbsbegin to atrophy from disuse. Muscle weakness becomes debilitating, andpatients eventually require a wheel chair or become confined to bed.Most ALS patients die from respiratory failure or from complications ofventilator assistance like pneumonia 3-5 years from disease onset.

In some embodiments, ALK4:ActRIIB heteromultimers of the disclosure maybe used to treat inflammatory muscle diseases/disorders such asdermatomyositis, inclusion body myositis or polymyositis. In someembodiments, the inflammatory muscle disease/disorder isdermatomyositis. In some embodiments, the inflammatory muscledisease/disorder is polymyositis. In some embodiments, the inflammatorymuscle disease/disorder is inclusion body myositis. In some embodiments,the inclusion body myositis is hereditary inclusion body myositis. Insome embodiments, the inclusion body myositis is sporadic inclusion bodymyositis.

Sporadic inclusion body myositis is associated with both autoimmune anddegenerative processes. This disorder typically first appears inpatients who are over 50 years of age, and predominantly appears inmales. Clinically, sporadic inclusion body myositis is characterized byprogressive quadriceps femoris and deep flexors weakness and atrophy.Many patients become wheelchair dependent and severely disabled 10-15years after symptom onset. Patients also frequently display dysphagiadue to esophageal and pharyngeal muscle involvement. Diagnosis forsporadic inclusion body myositis is usually based on some combination offactors such as elevated creatine kinase levels in the blood, abnormalelectromyography results; or muscle biopsies displaying inflammatorycell invasion of muscle, vacuolar degeneration and/or inclusions ofplaques of abnormal proteins.

Promotion of increased muscle mass by ALK4:ActRIIB heteromultimers mightalso benefit those suffering from muscle wasting diseases.Gonzalez-Cadavid et al. (supra) reported that GDF8 expression correlatesinversely with fat-free mass in humans and that increased expression ofthe GDF8 gene is associated with weight loss in men with AIDS wastingsyndrome. By inhibiting the function of GDF8 in AIDS patients, at leastcertain symptoms of AIDS may be alleviated, if not completelyeliminated, thus significantly improving quality of life in AIDSpatients.

Since loss of GDF8 function is also associated with fat loss withoutdiminution of nutrient intake (Zimmers et al., supra; McPherron and Lee,supra), the subject ALK4:ActRIIB heteromultimera may further be used asa therapeutic agent for slowing or preventing the development of obesityand type 2 diabetes.

Cancer anorexia-cachexia syndrome is among the most debilitating andlife-threatening aspects of cancer. This syndrome is a common feature ofmany types of cancer—present in approximately 80% of cancer patients atdeath—and is responsible not only for a poor quality of life and poorresponse to chemotherapy but also a shorter survival time than is foundin patients with comparable tumors but without weight loss. Cachexia istypically suspected in patients with cancer if an involuntary weightloss of greater than five percent of premorbid weight occurs within asix-month period. Associated with anorexia, wasting of fat and muscletissue, and psychological distress, cachexia arises from a complexinteraction between the cancer and the host. Cancer cachexia affectscytokine production, release of lipid-mobilizing andproteolysis-inducing factors, and alterations in intermediarymetabolism. Although anorexia is common, a decreased food intake aloneis unable to account for the changes in body composition seen in cancerpatients, and increasing nutrient intake is unable to reverse thewasting syndrome. Currently, there is no treatment to control or reversethe cachexic process. Since systemic overexpression of GDF8 in adultmice was found to induce profound muscle and fat loss analogous to thatseen in human cachexia syndromes (Zimmers et al., supra), the subjectALK4:ActRIIB heteromultimers may be beneficially used to prevent, treat,or alleviate the symptoms of the cachexia syndrome, where muscle growthis desired. An example of a heteromeric complex useful for preventing,treating, or alleviating muscle loss as described above is anALK4:ActRIIB heterodimer.

In certain embodiments, an ALK4:ActRIIB heteromultimer of the presentdisclosure may be used in methods of inducing bone and/or cartilageformation, preventing bone loss, increasing bone mineralization,preventing the demineralization of bone, and/or increasing bone density.ALK4:ActRIIB heteromultimers may be useful in patients who are diagnosedwith subclinical low bone density, as a protective measure against thedevelopment of osteoporosis.

In some embodiments, an ALK4:ActRIIB heteromultimer of the presentdisclosure may find medical utility in the healing of bone fractures andcartilage defects in humans and other animals. The subject methods andcompositions may also have prophylactic use in closed as well as openfracture reduction and also in the improved fixation of artificialjoints. De novo bone formation induced by an osteogenic agent is usefulfor repair of craniofacial defects that are congenital, trauma-induced,or caused by oncologic resection, and is also useful in cosmetic plasticsurgery. Further, methods and compositions of the invention may be usedin the treatment of periodontal disease and in other tooth repairprocesses. In certain cases, an ALK4:ActRIIB heteromultimer may providean environment to attract bone-forming cells, stimulate growth ofbone-forming cells, or induce differentiation of progenitors ofbone-forming cells. An ALK4:ActRIIB heteromultimer of the disclosure mayalso be useful in the treatment of osteoporosis. Further, ALK4:ActRIIBheteromultimers may be used in repair of cartilage defects andprevention/reversal of osteoarthritis. Examples of heteromeric complexesuseful for inducing bone formation, preventing bone loss, increasingbone mineralization, preventing the demineralization of bone, and/orincreasing bone density as described herein are ALK4:ActRIIBheterodimers.

Rosen et al. (ed) Primer on the Metabolic Bone Diseases and Disorders ofMineral Metabolism, 7^(th) ed. American Society for Bone and MineralResearch, Washington D.C. (incorporated herein by reference) provides anextensive discussion of bone disorders that may be subject to treatmentwith an ALK4:ActRIIB heteromultimer. A partial listing is providedherein. Methods and compositions of the invention can be applied toconditions characterized by or causing bone loss, such as osteoporosis(including secondary osteoporosis), hyperparathyroidism, chronic kidneydisease mineral bone disorder, sex hormone deprivation or ablation (e.g.androgen and/or estrogen), glucocorticoid treatment, rheumatoidarthritis, severe burns, hyperparathyroidism, hypercalcemia,hypocalcemia, hypophosphatemia, osteomalacia (including tumor-inducedosteomalacia), hyperphosphatemia, vitamin D deficiency,hyperparathyroidism (including familial hyperparathyroidism) andpseudohypoparathyroidism, tumor metastases to bone, bone loss as aconsequence of a tumor or chemotherapy, tumors of the bone and bonemarrow (e.g., multiple myeloma), ischemic bone disorders, periodontaldisease and oral bone loss, Cushing's disease, Paget's disease,thyrotoxicosis, chronic diarrheal state or malabsorption, renal tubularacidosis, or anorexia nervosa. Methods and compositions of the inventionmay also be applied to conditions characterized by a failure of boneformation or healing, including non-union fractures, fractures that areotherwise slow to heal, fetal and neonatal bone dysplasias (e.g.,hypocalcemia, hypercalcemia, calcium receptor defects and vitamin Ddeficiency), osteonecrosis (including osteonecrosis of the jaw) andosteogenesis imperfecta. Additionally, the anabolic effects will causesuch antagonists to diminish bone pain associated with bone damage orerosion. As a consequence of the anti-resorptive effects, suchantagonists may be useful to treat disorders of abnormal bone formation,such as osteoblastic tumor metastases (e.g., associated with primaryprostate or breast cancer), osteogenic osteosarcoma, osteopetrosis,progressive diaphyseal dysplasia, endosteal hyperostosis,osteopoikilosis, and melorheostosis. Other disorders that may be treatedinclude fibrous dysplasia and chondrodysplasias.

In another specific embodiment, the disclosure provides a therapeuticmethod and composition for repairing fractures and other conditionsrelated to cartilage and/or bone defects or periodontal diseases. Theinvention further provides therapeutic methods and compositions forwound healing and tissue repair. The types of wounds include, but arenot limited to, burns, incisions and ulcers. See, e.g., PCT PublicationNo. WO 84/01106. Such compositions comprise a therapeutically effectiveamount of at least one of the ALK4:ActRIIB heteromultimers of thedisclosure in admixture with a pharmaceutically acceptable vehicle,carrier, or matrix.

In some embodiments, an ALK4:ActRIIB heteromultimer of the disclosurecan be applied to conditions causing bone loss such as osteoporosis,hyperparathyroidism, Cushing's disease, thyrotoxicosis, chronicdiarrheal state or malabsorption, renal tubular acidosis, or anorexianervosa. It is commonly appreciated that being female, having a low bodyweight, and leading a sedentary lifestyle are risk factors forosteoporosis (loss of bone mineral density, leading to fracture risk).However, osteoporosis can also result from the long-term use of certainmedications. Osteoporosis resulting from drugs or another medicalcondition is known as secondary osteoporosis. In Cushing's disease, theexcess amount of cortisol produced by the body results in osteoporosisand fractures. The most common medications associated with secondaryosteoporosis are the corticosteroids, a class of drugs that act likecortisol, a hormone produced naturally by the adrenal glands. Althoughadequate levels of thyroid hormones are needed for the development ofthe skeleton, excess thyroid hormone can decrease bone mass over time.Antacids that contain aluminum can lead to bone loss when taken in highdoses by people with kidney problems, particularly those undergoingdialysis. Other medications that can cause secondary osteoporosisinclude phenytoin (Dilantin) and barbiturates that are used to preventseizures; methotrexate (Rheumatrex, Immunex, Folex PFS), a drug for someforms of arthritis, cancer, and immune disorders; cyclosporine(Sandimmune, Neoral), a drug used to treat some autoimmune diseases andto suppress the immune system in organ transplant patients; luteinizinghormone-releasing hormone agonists (Lupron, Zoladex), used to treatprostate cancer and endometriosis; heparin (Calciparine, Liquaemin), ananticlotting medication; and cholestyramine (Questran) and colestipol(Colestid), used to treat high cholesterol. Bone loss resulting fromcancer therapy is widely recognized and termed cancer therapy-inducedbone loss (CTIBL). Bone metastases can create cavities in the bone thatmay be corrected by treatment with an ALK4:ActRIIB heteromultimers. Boneloss can also be caused by gum disease, a chronic infection in whichbacteria located in gum recesses produce toxins and harmful enzymes.

In a further embodiment, the present disclosure provides methods andtherapeutic agents for treating diseases or disorders associated withabnormal or unwanted bone growth. For example, patients with thecongenital disorder fibrodysplasia ossificans progressiva (FOP) areafflicted by progressive ectopic bone growth in soft tissuesspontaneously or in response to tissue trauma, with a major impact onquality of life. Additionally, abnormal bone growth can occur after hipreplacement surgery and thus ruin the surgical outcome. This is a morecommon example of pathological bone growth and a situation in which thesubject methods and compositions may be therapeutically useful. The samemethods and compositions may also be useful for treating other forms ofabnormal bone growth (e.g., pathological growth of bone followingtrauma, burns or spinal cord injury), and for treating or preventing theundesirable conditions associated with the abnormal bone growth seen inconnection with metastatic prostate cancer or osteosarcoma.

In certain embodiments, an ALK4:ActRIIB heteromultimer of the disclosuremay be used to promote bone formation in patients with cancer. Patientshaving certain tumors (e.g. prostate, breast, multiple myeloma or anytumor causing hyperparathyroidism) are at high risk for bone loss due totumor-induced bone loss, bone metastases, and therapeutic agents. Suchpatients may be treated with a TGF-beta superfamily heteromultimercomplex, or a combination of complexes, even in the absence of evidenceof bone loss or bone metastases. Patients may also be monitored forevidence of bone loss or bone metastases, and may be treated with anALK4:ActRIIB heteromultimer in the event that indicators suggest anincreased risk. Generally, DEXA scans are employed to assess changes inbone density, while indicators of bone remodeling may be used to assessthe likelihood of bone metastases. Serum markers may be monitored. Bonespecific alkaline phosphatase (BSAP) is an enzyme that is present inosteoblasts. Blood levels of BSAP are increased in patients with bonemetastasis and other conditions that result in increased boneremodeling. Osteocalcin and procollagen peptides are also associatedwith bone formation and bone metastases. Increases in BSAP have beendetected in patients with bone metastasis caused by prostate cancer, andto a lesser degree, in bone metastases from breast cancer. BMP7 levelsare high in prostate cancer that has metastasized to bone, but not inbone metastases due to bladder, skin, liver, or lung cancer. Type Icarboxy-terminal telopeptide (ICTP) is a crosslink found in collagenthat is formed during to the resorption of bone. Since bone isconstantly being broken down and reformed, ICTP will be found throughoutthe body. However, at the site of bone metastasis, the level will besignificantly higher than in an area of normal bone. ICTP has been foundin high levels in bone metastasis due to prostate, lung, and breastcancer. Another collagen crosslink, Type I N-terminal telopeptide (NTx),is produced along with ICTP during bone turnover. The amount of NTx isincreased in bone metastasis caused by many different types of cancerincluding lung, prostate, and breast cancer. Also, the levels of NTxincrease with the progression of the bone metastasis. Therefore, thismarker can be used to both detect metastasis as well as measure theextent of the disease. Other markers of resorption include pyridinolineand deoxypyridinoline. Any increase in resorption markers or markers ofbone metastases indicate the need for therapy with an ALK4:ActRIIBheteromultimer in a patient.

In another embodiment, an ALK4:ActRIIB heteromultimer may be used inpatients with chronic kidney disease mineral bone disorder (CKD-MBD), abroad syndrome of interrelated skeletal, cardiovascular, andmineral-metabolic disorders arising from kidney disease. CKD-MBDencompasses various skeletal pathologies often referred to as renalosteodystrophy (ROD), which is a preferred embodiment for treatmentwith, an ALK4:ActRIIB heteromultimer. Depending on the relativecontribution of different pathogenic factors, ROD is manifested asdiverse pathologic patterns of bone remodeling (Hruska et al., 2008,Chronic kidney disease mineral bone disorder (CKD-MBD); in Rosen et al.(ed) Primer on the Metabolic Bone Diseases and Disorders of MineralMetabolism, 7th ed. American Society for Bone and Mineral Research,Washington D.C., pp 343-349). At one end of the spectrum is ROD withuremic osteodystrophy and low bone turnover, characterized by a lownumber of active remodeling sites, profoundly suppressed bone formation,and low bone resorption. At the other extreme is ROD withhyperparathyroidism, high bone turnover, and osteitis fibrosa. Giventhat an ALK4:ActRIIB heteromultimer may exert both anabolic andantiresorptive effects, these agents may be useful in patients acrossthe ROD pathology spectrum.

An ALK4:ActRIIB heteromultimer of the disclosure may be conjointlyadministered with other bone-active pharmaceutical agents. Conjointadministration may be accomplished by administration of a singleco-formulation, by simultaneous administration, or by administration atseparate times. ALK4:ActRIIB heteromultimers may be particularlyadvantageous if administered with other bone-active agents. A patientmay benefit from conjointly receiving an ALK4:ActRIIB heteromultimer andtaking calcium supplements, vitamin D, appropriate exercise and/or, insome cases, other medication. Examples of other medications include,bisphosphonates (alendronate, ibandronate and risedronate), calcitonin,estrogens, parathyroid hormone and raloxifene. The bisphosphonates(alendronate, ibandronate and risedronate), calcitonin, estrogens andraloxifene affect the bone remodeling cycle and are classified asanti-resorptive medications. Bone remodeling consists of two distinctstages: bone resorption and bone formation. Anti-resorptive medicationsslow or stop the bone-resorbing portion of the bone-remodeling cycle butdo not slow the bone-forming portion of the cycle. As a result, newformation continues at a greater rate than bone resorption, and bonedensity may increase over time. Teriparatide, a form of parathyroidhormone, increases the rate of bone formation in the bone remodelingcycle. Alendronate is approved for both the prevention (5 mg per day or35 mg once a week) and treatment (10 mg per day or 70 mg once a week) ofpostmenopausal osteoporosis. Alendronate reduces bone loss, increasesbone density and reduces the risk of spine, wrist and hip fractures.Alendronate also is approved for treatment of glucocorticoid-inducedosteoporosis in men and women as a result of long-term use of thesemedications (i.e., prednisone and cortisone) and for the treatment ofosteoporosis in men. Alendronate plus vitamin D is approved for thetreatment of osteoporosis in postmenopausal women (70 mg once a weekplus vitamin D), and for treatment to improve bone mass in men withosteoporosis. Ibandronate is approved for the prevention and treatmentof postmenopausal osteoporosis. Taken as a once-a-month pill (150 mg),ibandronate should be taken on the same day each month. Ibandronatereduces bone loss, increases bone density and reduces the risk of spinefractures. Risedronate is approved for the prevention and treatment ofpostmenopausal osteoporosis. Taken daily (5 mg dose) or weekly (35 mgdose or 35 mg dose with calcium), risedronate slows bone loss, increasesbone density and reduces the risk of spine and non-spine fractures.Risedronate also is approved for use by men and women to prevent and/ortreat glucocorticoid-induced osteoporosis that results from long-termuse of these medications (i.e., prednisone or cortisone). Calcitonin isa naturally occurring hormone involved in calcium regulation and bonemetabolism. In women who are more than 5 years beyond menopause,calcitonin slows bone loss, increases spinal bone density, and mayrelieve the pain associated with bone fractures. Calcitonin reduces therisk of spinal fractures. Calcitonin is available as an injection(50-100 IU daily) or nasal spray (200 IU daily).

A patient may also benefit from conjointly receiving an ALK4:ActRIIBheteromultimer and additional bone-active medications. Estrogen therapy(ET)/hormone therapy (HT) is approved for the prevention ofosteoporosis. ET has been shown to reduce bone loss, increase bonedensity in both the spine and hip, and reduce the risk of hip and spinalfractures in postmenopausal women. ET is administered most commonly inthe form of a pill or skin patch that delivers a low dose ofapproximately 0.3 mg daily or a standard dose of approximately 0.625 mgdaily and is effective even when started after age 70. When estrogen istaken alone, it can increase a woman's risk of developing cancer of theuterine lining (endometrial cancer). To eliminate this risk, healthcareproviders prescribe the hormone progestin in combination with estrogen(hormone replacement therapy or HT) for those women who have an intactuterus. ET/HT relieves menopause symptoms and has been shown to have abeneficial effect on bone health. Side effects may include vaginalbleeding, breast tenderness, mood disturbances and gallbladder disease.Raloxifene, 60 mg a day, is approved for the prevention and treatment ofpostmenopausal osteoporosis. It is from a class of drugs calledSelective Estrogen Receptor Modulators (SERMs) that have been developedto provide the beneficial effects of estrogens without their potentialdisadvantages. Raloxifene increases bone mass and reduces the risk ofspine fractures. Data are not yet available to demonstrate thatraloxifene can reduce the risk of hip and other non-spine fractures.Teriparatide, a form of parathyroid hormone, is approved for thetreatment of osteoporosis in postmenopausal women and men who are athigh risk for a fracture. This medication stimulates new bone formationand significantly increases bone mineral density. In postmenopausalwomen, fracture reduction was noted in the spine, hip, foot, ribs andwrist. In men, fracture reduction was noted in the spine, but there wereinsufficient data to evaluate fracture reduction at other sites.Teriparatide is self-administered as a daily injection for up to 24months.

In other embodiments, an ALK4:ActRIIB heteromultimer can be used forregulating body fat content in an animal and for treating or preventingconditions related thereto, and particularly, health-compromisingconditions related thereto. According to the present invention, toregulate (control) body weight can refer to reducing or increasing bodyweight, reducing or increasing the rate of weight gain, or increasing orreducing the rate of weight loss, and also includes activelymaintaining, or not significantly changing body weight (e.g., againstexternal or internal influences which may otherwise increase or decreasebody weight). One embodiment of the present disclosure relates toregulating body weight by administering to an animal (e.g., a human) inneed thereof a ALK4:ActRIIB heteromultimers.

In some embodiments, an ALK4:ActRIIB heteromultimer of the presentdisclosure can be used for reducing body weight and/or reducing weightgain in an animal, and more particularly, for treating or amelioratingobesity in patients at risk for or suffering from obesity. In anotherspecific embodiment, the present invention is directed to methods andcompounds for treating an animal that is unable to gain or retain weight(e.g., an animal with a wasting syndrome). Such methods are effective toincrease body weight and/or mass, or to reduce weight and/or mass loss,or to improve conditions associated with or caused by undesirably low(e.g., unhealthy) body weight and/or mass. In addition, disorders ofhigh cholesterol (e.g., hypercholesterolemia or dislipidemia) may betreated with an ALK4:ActRIIB heteromultimer of the disclosure.

In other embodiments, an ALK4:ActRIIB heteromultimer can be used forregulating body fat content in an animal and for treating or preventingconditions related thereto, and particularly, health-compromisingconditions related thereto. According to the present invention, toregulate (control) body weight can refer to reducing or increasing bodyweight, reducing or increasing the rate of weight gain, or increasing orreducing the rate of weight loss, and also includes activelymaintaining, or not significantly changing body weight (e.g., againstexternal or internal influences which may otherwise increase or decreasebody weight). One embodiment of the present disclosure relates toregulating body weight by administering to an animal (e.g., a human) inneed thereof an ALK4:ActRIIB heteromultimer. For example, in someembodiments, an ALK4:ActRIIB heteromultimer may be used to treat orprevent a disorder or condition selected from obesity (e.g., abdominalobesity); overweight; insulin resistance; metabolic syndrome and othermetabolic diseases or conditions; a lipid disorder such as, low HDLlevels, high LDL levels, hyperlipidemia, hypertriglyceridemia ordyslipidemia; lipoprotein aberrations; decreased triglycerides;inflammation (e.g., liver inflammation and/or inflammation of adiposetissue), fatty liver disease; non-alcoholic fatty liver disease;hyperglycemia; impaired glucose tolerance (IGT); hyperinsulinemia; highcholesterol (e.g., high LDL levels and hypercholesterolemia);cardiovascular disease such as, heart disease including coronary heartdisease, congestive heart failure, stroke, peripheral vascular disease,atherosclerosis; arteriosclerosis, and hypertension; Syndrome X;vascular restenosis; neuropathy; retinopathy; neurodegenerative disease;endothelial dysfunction, respiratory dysfunction; pancreatitis;polycystic ovarian syndrome; elevated uric acid levels; haemochromatosis(iron overload); acanthosis nigricans (dark patches on the skin); orcancer (e.g., ovarian, breast, endometrial, and colon cancer); or aanother disorders/conditions associated with one or more of the abovediseases or conditions. In some embodiments, the disease or conditiontreated using an ALK4:ActRIIB heteromultimer is associated withoverweight (e.g., BMI of ≥25 kg/m²), or with too much body fat.

In one embodiment, the disclosure provides a method of reducing bodyweight comprising administering to a subject desiring to reduce bodyweight, or in need thereof, an effective amount of an ALK4:ActRIIBheteromultimer. In some embodiments, the subject is overweight (e.g.,pre-obese). In some embodiments, the subject has a body mass index (BMI)of 25 kg/m² or greater. In further embodiments, the subject has a BMI of25 kg/m² to 29.9 kg/m², 30 kg/m² to 39.9 kg/m², 25 kg/m² to 39.9 kg/m²,or 25 kg/m² to 50 kg/m². In some embodiments, the subject is obese. Insome embodiments, the subject has a BMI of 30 kg/m² or greater (e.g., 30to 39.9 kg/m² or 30 kg/m² to 50 kg/m²). In some embodiments, the subjectis morbidly obese. In some embodiments, the subject has a BMI of 40kg/m² or greater. In further embodiments, the subject has a BMI of 40kg/m² to 45 kg/m², or 40 kg/m² to 50 kg/m². In some embodiments, thesubject has central obesity (e.g., excess adiposity in the abdominalregion, including belly fat and/or visceral fat). In some embodiments,the subject has a waist/hip circumference ratio (WHR) of 0.85 orgreater. In some embodiments, the subject has peripheral obesity (e.g.,excess adiposity on the hips). In some embodiments, the subject has type2 diabetes mellitus. The ALK4:ActRIIB heteromultimer may administeredalone or as a combination therapy other type of supportive therapy. Forexample, in some embodiments, the supportive therapy is diet and/orexercise.

In one embodiment, the disclosure provides a method of reducing weightgain comprising administering to a subject desiring to reduce weightgain, or in need thereof, an effective amount of an ALK4:ActRIIBheteromultimer. In some embodiments, the subject is overweight (e.g.,pre-obese). In some embodiments, the subject has a BMI of 25 kg/m² orgreater. In further embodiments, the subject has a BMI of 25 kg/m² to29.9 kg/m², 30 kg/m² to 39.9 kg/m², 25 kg/m² to 39.9 kg/m², or 25 kg/m²to 50 kg/m². In some embodiments, the subject is obese. In someembodiments, the subject has a BMI of 30 kg/m² or greater (e.g., 30 to39.9 kg/m² or 30 kg/m² to 50 kg/m²). In some embodiments, the subject ismorbidly obese. In some embodiments, the subject has a BMI of 40 kg/m²or greater. In further embodiments, the subject has a BMI of 40 kg/m² to45 kg/m², or 40 kg/m² to 50 kg/m². In some embodiments, the subject hastype 2 diabetes mellitus.

Also provided is a method of treating or preventing a disease orcondition associated with excess body weight, comprising administeringto a subject in need of treatment or prevention, an effective amount ofan ALK4:ActRIIB heteromultimer. In one embodiment, the treated orprevented disease or condition is obesity. In one embodiment, thetreated or prevented disease or condition is insulin resistance. In oneembodiment, the treated or prevented disease or condition is a memberselected from the group consisting of: dyslipidemia, hyperlipidemia(total cholesterol level >240 mg/dL), hypercholesterolemia (e.g., totalcholesterol level of >200 mg/dL, >220 mg/dL, >240 mg/dL, >250 mg/dL,or >275 mg/dL), low HDL serum level (e.g., <40 mg/dL, <45 mg/dL, or <50mg/dL), high LDL serum level (e.g., ≥100 mg/dL, ≥130 mg/dL, ≥160 mg/dL,or ≥190 mg/dL), and hypertriglyceridemia (e.g., a fasting TG level of≥150 mg/dL, ≥175 mg/dL, ≥200 mg/dL, ≥300 mg/dL, ≥400 mg/dL, or ≥499mg/dL). In certain instances, the ALK4:ActRIIB antagonists treatment isan adjunct to diet and/or exercise.

In another embodiment the disclosure provides a method of reducing bodyweight in a subject who is overweight. The method includes administeringto an overweight subject an effective amount of an ALK4:ActRIIBheteromultimer. In some embodiments, the subject has a body mass index(BMI) of 25 kg/m² or greater. In further embodiments, the subject has aBMI of 25 kg/m² to 29.9 kg/m², 30 kg/m² to 39.9 kg/m², 25 kg/m² to 39.9kg/m², or 25 kg/m² to 50 kg/m²′ or 27 to 40 kg/m². In some embodiments,the subject is obese. In some embodiments, the subject has a BMI of 30kg/m² or greater (e.g., 30 to 39.9 kg/m² or 30 kg/m² to 50 kg/m²). TheALK4:ActRIIB heteromultimer is administered alone or as a combinationtherapy. In some embodiments, the ALK4:ActRIIB heteromultimer treatmentis an adjunct to diet and/or exercise.

In one embodiment the disclosure provides a method of reducing bodyweight in an obese subject. The method includes administering to thesubject an effective amount of an ALK4:ActRIIB heteromultimer. In someembodiments, the subject has a BMI of 30 kg/m² or greater (e.g., 30 to39.9 kg/m² or 30 kg/m² to 50 kg/m^(2.) In some embodiments, the subjecthas a BMI of 40 kg/m² or greater. In some embodiments, the subject hascentral obesity (e.g., excess adiposity in the abdominal region,including belly fat and/or visceral fat). In some embodiments, thesubject has a waist/hip circumference ratio (WHR) of 0.85 or greater. Insome embodiments, the subject has peripheral obesity (e.g., excessadiposity on the hips). In some embodiments, the ALK4:ActRIIBheteromultimer treatment is an adjunct to diet and/or exercise.

In another embodiment, the disclosure provides a method of treatingand/or ameliorating obesity or a disease or condition associated withobesity, comprising administering to an obese subject, an effectiveamount of an ALK4:ActRIIB heteromultimer. In some embodiments, thesubject has a BMI of 30 kg/m² or greater. In further embodiments, thesubject has a BMI of 30 to 39.9 kg/m² or 30 kg/m² to 50 kg/m². In someembodiments, the subject is morbidly obese. In some embodiments, thesubject has a body BMI of 40 kg/m² or greater. In further embodiments,the subject has a BMI of 40 kg/m² to 45 kg/m², or 40 kg/m² to 50 kg/m²Insome embodiments, the subject has type 2 diabetes mellitus. In someembodiments, the subject has a BMI of 30 kg/m² or greater (e.g., 30 to39.9 kg/m²). In some embodiments, the subject has a BMI of at least 40kg/m². In some embodiments, the subject has central obesity (e.g.,excess adiposity in the abdominal region, including belly fat and/orvisceral fat). In some embodiments, the subject has a waist/hipcircumference ratio (WHR) of 0.85 or greater. In some embodiments, thesubject has peripheral obesity (e.g., excess adiposity on the hips). Insome embodiments, the ALK4:ActRIIB heteromultimer treatment is anadjunct to diet and/or exercise.

Also provided is a method of treating or preventing a disease orcondition associated with obesity, comprising administering to a subjectin need of treatment or prevention, an effective amount of anALK4:ActRIIB heteromultimer. In one embodiment, the treated or preventeddisease or condition is a member selected from the group consisting of:dyslipidemia, hyperlipidemia (total cholesterol level >240 mg/dL),hypercholesterolemia (e.g., total cholesterol level of >200 mg/dL, >220mg/dL, >240 mg/dL, >250 mg/dL, or >275 mg/dL), low HDL serum level(e.g., <40 mg/dL, <45 mg/dL, or <50 mg/dL), high LDL serum level (e.g.,≥100 mg/dL, ≥130 mg/dL, ≥160 mg/dL, or ≥190 mg/dL), andhypertriglyceridemia (e.g., a fasting TG level of ≥150 mg/dL, ≥175mg/dL, ≥200 mg/dL, ≥300 mg/dL, ≥400 mg/dL, or ≥499 mg/dL). In oneembodiment, the treated or prevented disease or condition iscardiovascular disease. In an additional embodiment, the treated orprevented disease or condition is hypertension (high blood pressure),myocardial infarction, peripheral artery disease, vasoregulatoindysfunction, arteriosclerosis congestive heart failure, atherosclerosis,coronary heart disease, or microvascular disease. In one embodiment, thetreated or prevented disease or condition is liver disease. In oneembodiment, the treated or prevented liver disease or condition isNAFLD. In one embodiment, the liver disease is fatty liver. In oneembodiment, the liver disease is NASH. In another embodiment, thetreated or prevented disease or condition is a member selected from thegroup: steatohepatitis, steatosis, fibrosis, and/or cirrhosis. Incertain instances, the ALK4:ActRIIB heteromultimer treatment is anadjunct to diet and/or exercise.

In another embodiment, the disclosure provides a method of treating,ameliorating, and/or preventing type 2 diabetes mellitus or a disease orcondition associated with diabetes comprising administering to a subjecthaving type 2 diabetes mellitus, or at risk of developing type 2diabetes, an effective amount of an heteromultimer. In some embodiments,the subject has a body mass index BMI of 30 kg/m² or greater (e.g., 30to 39.9 kg/m²). In some embodiments, the subject has a BMI of at least40 kg/m². In some embodiments, the subject has central obesity (e.g.,excess adiposity in the abdominal region, including belly fat and/orvisceral fat). In some embodiments, the subject has a WHR of 0.85 orgreater. In some embodiments, the subject has peripheral obesity (e.g.,excess adiposity on the hips). In some embodiments, the ALK4:ActRIIBheteromultimer treatment is an adjunct to diet and/or exercise.

Also provided is a method of treating, ameliorating or preventing adisease or condition associated with diabetes, comprising administeringto a subject having diabetes, an effective amount of an ALK4:ActRIIBheteromultimer. In one embodiment, the treated or prevented disease orcondition is a member selected from the group consisting of:dyslipidemia, hyperlipidemia (total cholesterol level >240 mg/dL),hypercholesterolemia (e.g., total cholesterol level of >200 mg/dL, >220mg/dL, >240 mg/dL, >250 mg/dL, or >275 mg/dL), low HDL serum level(e.g., <40 mg/dL, <45 mg/dL, or <50 mg/dL), high LDL serum level (e.g.,≥100 mg/dL, ≥130 mg/dL, ≥160 mg/dL, or ≥190 mg/dL), andhypertriglyceridemia (e.g., a fasting TG level of ≥150 mg/dL, ≥175mg/dL, ≥200 mg/dL, ≥300 mg/dL, ≥400 mg/dL, or ≥499 mg/dL). In oneembodiment, the treated or prevented disease or condition iscardiovascular disease. In an additional embodiment, the treated orprevented disease or condition is hypertension (high blood pressure),myocardial infarction, peripheral artery disease, vasoregulatoindysfunction, or arteriosclerosis. In one embodiment, the treated orprevented disease or condition is liver disease. In another embodiment,the treated or prevented disease or condition is a member selected fromthe group: fatty liver disease, steatohepatitis, steatosis, and/orcirrhosis. In one embodiment, the treated or prevented disease orcondition is a member selected from the group consisting of: cataracts,obstructive sleep apnea, phlebitis, gout, osteoarthritis, gallbladderdisease, and high cholesterol. In certain instances, the ALK4:ActRIIBheteromultimer treatment is an adjunct to diet and/or exercise.

The disclosure also provides a method for improving the blood-lipidprofile in a subject, comprising administering to a subject in need ofsuch treatment an effective amount of an ALK4:ActRIIB heteromultimer. Insome embodiments, the disclosure provides a method for reducing levelsof LDL cholesterol or increasing levels of HDL-cholesterol. In oneembodiment, the subject has dyslipidemia. In another embodiment, thesubject has elevated serum lipids (e.g., cholesterol(hypercholesterolemia) and/or triglycerides (e.g.,hypertriglyceridemia). In one embodiment the subject has an LDL-C ≥100mg/dL, ≥130 mg/dL, or ≥160 mg/dL). In one embodiment the subject has aTG ≥150 mg/dL, ≥160 mg/dL, ≥170 mg/dL). In one embodiment, the subjecthas elevated plasma insulin levels (hyperinsulinemia; e.g., fastinginsulin level of >20 ug/ml can exceed 100). In some embodiments, thesubject has type II diabetes.

According to one embodiment, the disclosure provides a method oftreating or preventing a metabolic disease or disorder or a conditionassociated with a metabolic disease or disorder, comprisingadministering an ALK4:ActRIIB heteromultimer to a subject in needthereof. In one embodiment, the treated metabolic disease, disorder, orcondition is hyperglycemia (e.g., >130 mg/dL in the fasting state orfollowing glucose administration during an oral glucose tolerance test).In one embodiment, the treated metabolic disease, disorder, or conditionis a lipid metabolism disease, disorder, or condition. In oneembodiment, the treated metabolic disease, disorder, or condition isdislipidemia. In a further embodiment, the lipid metabolism disease,disorder, or condition is a member selected from: low HDL levels, highLDL levels, high triglyceride levels, hyperlipidemia, and a lipoproteinaberration. In one embodiment, the subject has a total cholesterol levelof >200 mg/dL, >220 mg/dL, >240 mg/dL, >250 mg/dL, or >275 mg/dL. In oneembodiment, the subject has a HDL serum level of <40 mg/dL, <45 mg/dL,or <50 mg/dL). In one embodiment, the subject has a LDL serum level ≥100mg/dL, ≥130 mg/dL, ≥160 mg/dL, or ≥190 mg/dL. In one embodiment, thesubject has fasting TG level of ≥150 mg/dL, ≥175 mg/dL, ≥200 mg/dL, ≥300mg/dL, ≥400 mg/dL, or ≥499 mg/dL. In one embodiment, the treatedmetabolic disease, disorder, or condition is a glucose metabolismdisease, disorder, or condition. In a further embodiment, the glucosemetabolism disease, disorder, or condition is a member selected from:glucose intolerance, insulin resistance, impaired glucose tolerance(IGT), impaired fasting glucose (IFG). In one embodiment, the treatedmetabolic disease, disorder, or condition is a member selected from thegroup consisting of: high uric acid levels, NAFLD, fatty liver, NASH,and polycystic ovarian syndrome. In one embodiment, the treated subjecthas hyperinsulinemia. In one embodiment, the treated subject is obese(e.g., the subject has abdominal obesity). In another embodiment, thetreated subject has type II diabetes.

Metabolic syndrome is a condition involving a set of disorders thatenhances the risk of heart disease. The major components of metabolicsyndrome are excess weight, the cardiovascular parameters (high bloodpressure, dyslipidemia, high levels of triglycerides and/or low levelsof HDL in the blood), atherosclerosis, diabetes, and/or insulinresistance. A subject having several of these components, i.e. metabolicsyndrome, is highly prone to heart disease, though each component is arisk factor. The disclosure also provides a method for treating orpreventing 1, 2, 3, or more of the above components of metabolicsyndrome, comprising administering to a subject in need of treatment aneffective amount of an ALK4:ActRIIB heteromultimer.

Additionally provided is a method of treating, preventing orameliorating a cardiovascular disease or condition, comprisingadministering an ALK4:ActRIIB heteromultimer to a subject in needthereof. In one embodiment, the treated, prevented, or amelioratedcardiovascular disease or condition is atherosclerosis. In oneembodiment, the treated, prevented, or ameliorated cardiovasculardisease or condition is hypertension (e.g., blood pressure >130/80 mmHgor >140/90 mmHg, in a resting state. In one embodiment, thecardiovascular disease is atherosclerosis (coronary heart disease).

In one embodiment, the disclosure provides a method for treating and/orameliorating an inflammatory liver disease or condition that comprisesadministering an ALK4:ActRIIB heteromultimer, to a subject in needthereof. In one embodiment, the disease or condition is NAFLD. In afurther embodiment, the disease or condition is fatty liver. In afurther embodiment, the disease or condition is steatosis (e.g.,nonalcoholic steatohepatitis (NASH)). In a further embodiment, thedisease or condition is alcoholic fatty liver disease.

This disclosure also provides a method of improving glycemic control,comprising administering to a subject in need of treatment an effectiveamount of an ALK4:ActRIIB heteromultimer. In one embodiment, the subjectis administered has a fasting blood sugar levelof >130, >135, >140, >145, or >150 mg/dL. In one embodiment, the subjectis administered has a postprandial blood sugar levelof >180, >185, >190, >195, or >200 mg/dL 2 hours after eating. Incertain instances, the ALK4:ActRIIB heteromultimer treatment is anadjunct to diet and/or exercise. The administration can also reduce bodyweight or treat obesity. In certain instances, the subject has type 2diabetes mellitus. In certain instances, the subject has a BMI of 27 to40 kg/m2. In certain instances, the subject has a BMI of 30 to 39.9kg/m2. In certain instances, the subject has a BMI of at least 40. Incertain instances, the subject is overweight. In certain instances, thesubject is obese. An improvement in glycemic control can be assessedusing techniques known in the art such as a mixed-meal test.

The disclosure also provides compositions and methods for treating,preventing or ameliorating hyperglycemia or a condition associated withhyperglycemia in a subject comprising administering to a subject in needof such treatment an effective amount of an ALK4:ActRIIB heteromultimer.In one embodiment, the subject is administered has a fasting blood sugarlevel of >130, >135, >140, >145, or >150 mg/dL. In one embodiment, thesubject is administered has a postprandial blood sugar levelof >180, >185, >190, >195, or >200 mg/dL 2 hours after eating. In oneembodiment, the result of the treatment, prevention or amelioration is amember selected from the group consisting of: a decrease in serum levelsof glucose, a decrease in serum levels of triglycerides, a decrease inserum levels of insulin, and/or a decrease in serum levels ofnon-esterified fatty acids, as compared to serum levels in the subjectprior to treatment. In one embodiment, the result of the treatment,prevention or amelioration is an increase in body temperature of about0.4° C. to 1° C. as compared to body temperature of the subject prior totreatment. In some embodiments, the ALK4:ActRIIB treatment also reducesbody weight of the subject.

In another embodiment, the disclosure provides a method of decreasingplasma insulin levels in a subject, comprising administering aneffective amount of an ALK4:ActRIIB heteromultimer to a subject in needof such treatment. In one embodiment, the subject has a fasting bloodsugar level of >130, >135, >140, >145, or >150 mg/dL. In one embodiment,the subject has a postprandial blood sugar levelof >180, >185, >190, >195, or >200 mg/dL 2 hours after eating. In oneembodiment, the subject is overweight. In one embodiment, the subject isobese. In another embodiment, the subject has type 2 diabetes.

The disclosure also provides compositions and methods for treating,preventing or ameliorating hyperglycemia or a condition associated withhyperglycemia in a subject comprising administering to a subject in needof such treatment an effective amount of an ALK4:ActRIIB heteromultimer.In one embodiment, the subject has a fasting blood sugar levelof >130, >135, >140, >145, or >150 mg/dL. In one embodiment, the subjecthas a postprandial blood sugar level of >180, >185, >190, >195, or >200mg/dL 2 hours after eating. In one embodiment, the result of thetreatment, prevention or amelioration is a member selected from thegroup consisting of: a decrease in serum levels of glucose, a decreasein serum levels of triglycerides, a decrease in serum levels of insulin,and/or a decrease in serum levels of non-esterified fatty acids, ascompared to serum levels in the subject prior to treatment. In oneembodiment, the result of the treatment, prevention or amelioration isan increase in body temperature of about 0.4° C. to 1° C. as compared tobody temperature of the subject prior to treatment. In some embodiments,the ALK4:ActRIIB heteromultimer treatment also reduces body weight ofthe subject.

In another embodiment, the disclosure provides a method of decreasingplasma insulin levels in a subject, comprising administering aneffective amount of an ALK4:ActRIIB heteromultimer to a subject in needof such treatment. In one embodiment, the subject has a fasting bloodsugar level of >130, >135, >140, >145, or >150 mg/dL. In one embodiment,the has a postprandial blood sugar level of >180, >185, >190, >195,or >200 mg/dL 2 hours after eating. In one embodiment, the subject isoverweight. In one embodiment, the subject is obese. In anotherembodiment, the subject has type 2 diabetes. In another embodiment, thedisclosure provides a method of treating, preventing, or amelioratingliver disease in a subject, comprising administering an effective amountof an ALK4:ActRIIB heteromultimer to a subject having a liver disease.In one embodiment, the subject has inflammation of the liver. In oneembodiment, the subject has NAFLD. In on embodiment the subject hasfatty liver. In another embodiment, the subject has NASH. In onembodiment the subject has fatty liver. In another embodiment, thesubject has alcoholic fatty liver disease. In one embodiment, thetreated, prevented or ameliorated liver disease is fibrosis, scarring,cirrhosis, or liver failure. In another embodiment, the treated,prevented or ameliorated liver disease is liver cancer. In oneembodiment, the subject is overweight. In another embodiment, thesubject is obese. In another embodiment, the subject has type 2diabetes.

Fibrosis generally refers to an excessive deposition of both collagenfibers and extracellular matrix combined with a relative decrease ofcell number in an organ or tissue. While this process is an importantfeature of natural wound healing following injury, fibrosis can lead topathological damage in various tissue and organs including, for example,the lungs, kidneys, liver, bone, muscle, and skin. The role TGF-beta infibrosis has been extensively study. However, other TGF-beta superfamilyligands have also been implicated in fibrosis including, for example,activins (e.g., activin A and activin B) and GDF8 [Hedger et al (2013)Cytokine and Growth Factor Reviews 24:285-295; Hardy et al. (2015) 93:567-574; and Cantini et al. (2008) J Sex Med 5:1607-1622]. Therefore, insome embodiments, an ALK4:ActRIIB heteromultimer of the presentdisclosure can be used to treat fibrosis, particularlyfibrosis-associated disorders and conditions. For example, anALK4:ActRIIB heteromultimer may be used to treat or prevent one or moreof: pulmonary fibrosis, hypersensitivity pneumonitis, idiopathicfibrosis, tuberculosis, pneumonia, cystic fibrosis, asthma, chronicobstructive pulmonary disease (COPD), emphysema, renal (kidney)fibrosis, renal (kidney) failure, chronic renal (kidney) disease, bonefibrosis, myelofibrosis, rheumatoid arthritis, systemic lupuserythematosus, scleroderma, sarcoidosis, granulomatosis withpolyangiitis, Peyronie's disease, liver fibrosis, Wilson's disease,glycogen storage diseases (particularly types III, IV, IX, and X),iron-overload, Gaucher disease, Zellweger syndrome, nonalcoholic andalcoholic steatohepatitis, biliary cirrhosis, sclerosing cholangitis,Budd-Chiari syndrome, surgery-associated fibrosis, Crohn's disease,Duputren's contracture, mediastinal fibrosis, nephrogeneic fibrosis,retroperitoneal fibrosis, atrial fibrosis, endomyocardial fibrosis,pancreatic fibrosis and idiopathic pulmonary fibrosis.

The kidneys maintain many features of the blood, including volume, pHbalance, electrolyte concentrations, and blood pressure, as well asbearing responsibility for toxin and waste filtration. These functionsdepend upon the intricate structure of the kidney nephrons, constantflow of blood through the various capillaries of the kidney, and theregulation of the kidney by signals from the rest of the body, includingendocrine hormones. Problems with kidney function manifest by directmechanisms (e.g. genetic defects, infection, or toxin exposure) and byindirect mechanisms progressively proceeding from long term stressorslike hypertrophy and hyperfiltration (themselves often a result of moredirect insults to kidney function). Due to the central role of thekidney in blood maintenance and waste secretion, kidney-associateddisease manifestations are many and varied; they can be reviewed inHarrison's Principles of Internal Medicine, 18^(th) edition, McGrawHill, N.Y., Part 13, Chp 277-289.

As described herein, an ALK4:ActRIIB heteromultimer had variousbeneficial effects in a kidney disease model. In particular, treatmentwith an ALK4:ActRIIB heteromultimer reduced kidney tissue damage,inflammation, and fibrosis in subjects having unilateral ureteralobstruction. These data indicate that ALK4:ActRIIB heteromultimer may beused to treat or prevent kidney disease, particularly treating orpreventing various complications (manifestations) of kidney diseaseincluding, for example, kidney tissue damage, inflammation, and/orfibrosis.

Therefore, methods of this invention can be applied to variouskidney-associated diseases or conditions. As used herein,“kidney-associated disease or condition” can refer to any disease,disorder, or condition that affects the kidneys or the renal system.Examples of kidney-associated diseases or conditions include, but arenot limited to, chronic kidney diseases (or failure), acute kidneydiseases (or failure), primary kidney diseases, non-diabetic kidneydiseases, glomerulonephritis, interstitial nephritis, diabetic kidneydiseases, diabetic nephropathy, glomerulosclerosis, rapid progressiveglomerulonephritis, renal fibrosis, Alport syndrome, IDDM nephritis,mesangial proliferative glomerulonephritis, membranoproliferativeglomerulonephritis, crescentic glomerulonephritis, renal interstitialfibrosis, focal segmental glomerulosclerosis, membranous nephropathy,minimal change disease, pauci-immune rapid progressiveglomerulonephritis, IgA nephropathy, polycystic kidney disease, Dent'sdisease, nephrocytinosis, Heymann nephritis, autosomal dominant (adult)polycystic kidney disease, autosomal recessive (childhood) polycystickidney disease, acute kidney injury, nephrotic syndrome, renal ischemia,podocyte diseases or disorders, proteinuria, glomerular diseases,membranous glomerulonephritis, focal segmental glomerulonephritis,pre-eclampsia, eclampsia, kidney lesions, collagen vascular diseases,benign orthostatic (postural) proteinuria, IgM nephropathy, membranousnephropathy, sarcoidosis, diabetes mellitus, kidney damage due to drugs,Fabry's disease, aminoaciduria, Fanconi syndrome, hypertensivenephrosclerosis, interstitial nephritis, Sickle cell disease,hemoglobinuria, myoglobinuria, Wegener's Granulomatosis, GlycogenStorage Disease Type 1, chronic kidney disease, chronic renal failure,low Glomerular Filtration Rate (GFR), nephroangiosclerosis, lupusnephritis, ANCA-positive pauci-immune crescentic glomerulonephritis,chronic allograft nephropathy, nephrotoxicity, renal toxicity, kidneynecrosis, kidney damage, glomerular and tubular injury, kidneydysfunction, nephritic syndrome, acute renal failure, chronic renalfailure, proximal tubal dysfunction, acute kidney transplant rejection,chronic kidney transplant rejection, non-IgA mesangioproliferativeglomerulonephritis, postinfectious glomerulonephritis, vasculitides withrenal involvement of any kind, any hereditary renal disease, anyinterstitial nephritis, renal transplant failure, kidney cancer, kidneydisease associated with other conditions (e.g., hypertension, diabetes,and autoimmune disease), Dent's disease, nephrocytinosis, Heymannnephritis, a primary kidney disease, a collapsing glomerulopathy, adense deposit disease, a cryoglobulinemia-associated glomerulonephritis,an Henoch-Schonlein disease, a postinfectious glomerulonephritis, abacterial endocarditis, a microscopic polyangitis, a Churg-Strausssyndrome, an anti-GBM-antibody mediated glomerulonephritis, amyloidosis,a monoclonal immunoglobulin deposition disease, a fibrillaryglomerulonephritis, an immunotactoid glomerulopathy, ischemic tubularinjury, a medication-induced tubulo-interstitial nephritis, a toxictubulo-interstitial nephritis, an infectious tubulo-interstitialnephritis, a bacterial pyelonephritis, a viral infectioustubulo-interstitial nephritis which results from a polyomavirusinfection or an HIV infection, a metabolic-induced tubulo-interstitialdisease, a mixed connective disease, a cast nephropathy, a crystalnephropathy which may results from urate or oxalate or drug-inducedcrystal deposition, an acute cellular tubulo-interstitial allograftrejection, a tumoral infiltrative disease which results from a lymphomaor a post-transplant lymphoproliferative disease, an obstructive diseaseof the kidney, vascular disease, a thrombotic microangiopathy, anephroangiosclerosis, an atheroembolic disease, a mixed connectivetissue disease, a polyarteritis nodosa, a calcineurin-inhibitorinduced-vascular disease, an acute cellular vascular allograftrejection, an acute humoral allograft rejection, early renal functiondecline (ERFD), end stage renal disease (ESRD), renal vein thrombosis,acute tubular necrosis, acute interstitial nephritis, establishedchronic kidney disease, renal artery stenosis, ischemic nephropathy,uremia, drug and toxin-induced chronic tubulointerstitial nephritis,reflux nephropathy, kidney stones, Goodpasture's syndrome, normocyticnormochromic anemia, renal anemia, diabetic chronic kidney disease,IgG4-related disease, von Hippel-Lindau syndrome, tuberous sclerosis,nephronophthisis, medullary cystic kidney disease, renal cell carcinoma,adenocarcinoma, nephroblastoma, lymphoma, leukemia, hyposialylationdisorder, chronic cyclosporine nephropathy, renal reperfusion injury,renal dysplasia, azotemia, bilateral arterial occlusion, acute uric acidnephropathy, hypovolemia, acute bilateral obstructive uropathy,hypercalcemic nephropathy, hemolytic uremic syndrome, acute urinaryretention, malignant nephrosclerosis, postpartum glomerulosclerosis,scleroderma, non-Goodpasture's anti-GBM disease, microscopicpolyarteritis nodosa, allergic granulomatosis, acute radiationnephritis, post-streptococcal glomerulonephritis, Waldenstrom'smacroglobulinemia, analgesic nephropathy, arteriovenous fistula,arteriovenous graft, dialysis, ectopic kidney, medullary sponge kidney,renal osteodystrophy, solitary kidney, hydronephrosis, microalbuminuria,uremia, haematuria, hyperlipidemia, hypoalbuminaemia, lipiduria,acidosis, hyperkalemia, and edema.

In some embodiments, an ALK4:ActRIIB heteromultimer of the presentdisclosure may be used to treat or prevent chronic kidney disease,optionally in combination with one or more supportive therapies fortreating chronic kidney disease. In some embodiments, an ALK4:ActRIIBheteromultimer of the present disclosure may be used to treat or preventone or more complications (symptoms or manifestations) of chronic kidneydisease, optionally in combination with one or more supportive therapiesfor treating chronic kidney disease. In some embodiments, anALK4:ActRIIB heteromultimer of the present disclosure may be used totreat or prevent end-stage kidney failure, optionally in combinationwith one or more supportive therapies for treating end-stage kidneydisease. Chronic kidney disease (CKD), also known as chronic renaldisease, is a progressive loss in renal function over a period of monthsor years. The symptoms of worsening kidney function may include feelinggenerally unwell and experiencing a reduced appetite. Often, chronickidney disease is diagnosed as a result of screening of people known tobe at risk of kidney problems, such as those with high blood pressure ordiabetes and those with a blood relative with CKD. This disease may alsobe identified when it leads to one of its recognized complications, suchas cardiovascular disease, anemia, or pericarditis. Recent professionalguidelines classify the severity of CKD in five stages, with stage 1being the mildest and usually causing few symptoms and stage 5 being asevere illness with poor life expectancy if untreated. Stage 5 CKD isoften called end-stage kidney disease, end-stage renal disease, orend-stage kidney failure, and is largely synonymous with the nowoutdated terms chronic renal failure or chronic kidney failure; andusually means the patient requires renal replacement therapy, which mayinvolve a form of dialysis, but ideally constitutes a kidney transplant.CKD is initially without specific symptoms and is generally onlydetected as an increase in serum creatinine or protein in the urine. Asthe kidney function decreases and various symptoms may manifest asdescribed below. Blood pressure may be increased due to fluid overloadand production of vasoactive hormones created by the kidney via therenin-angiotensin system, increasing one's risk of developinghypertension and/or suffering from congestive heart failure. Urea mayaccumulate, leading to azotemia and ultimately uremia (symptoms rangingfrom lethargy to pericarditis and encephalopathy). Due to its highsystemic circulation, urea is excreted in eccrine sweat at highconcentrations and crystallizes on skin as the sweat evaporates (“uremicfrost”). Potassium may accumulate in the blood (hyperkalemia with arange of symptoms including malaise and potentially fatal cardiacarrhythmias). Hyperkalemia usually does not develop until the glomerularfiltration rate falls to less than 20-25 ml/min/1.73 m2, at which pointthe kidneys have decreased ability to excrete potassium. Hyperkalemia inCKD can be exacerbated by acidemia (which leads to extracellular shiftof potassium) and from lack of insulin. Erythropoietin synthesis may bedecreased causing anemia. Fluid volume overload symptoms may occur,ranging from mild edema to life-threatening pulmonary edema.Hyperphosphatemia, due to reduced phosphate excretion, may occurgenerally following the decrease in glomerular filtration.Hyperphosphatemia is associated with increased cardiovascular risk,being a direct stimulus to vascular calcification. Hypocalcemia maymanifest, which is generally caused by stimulation of fibroblast growthfactor-23. Osteocytes are responsible for the increased production ofFGF23, which is a potent inhibitor of the enzyme 1-alpha-hydroxylase(responsible for the conversion of 25-hydroxycholecalciferol into 1,25dihydroxyvitamin D3). Later, this progresses to secondaryhyperparathyroidism, renal osteodystrophy, and vascular calcificationthat further impairs cardiac function. Metabolic acidosis (due toaccumulation of sulfates, phosphates, uric acid etc.) may occur andcause altered enzyme activity by excess acid acting on enzymes; and alsoincreased excitability of cardiac and neuronal membranes by thepromotion of hyperkalemia due to excess acid (acidemia). Acidosis isalso due to decreased capacity to generate enough ammonia from the cellsof the proximal tubule. Iron deficiency anemia, which increases inprevalence as kidney function decreases, is especially prevalent inthose requiring haemodialysis. It is multifactoral in cause, butincludes increased inflammation, reduction in erythropoietin, andhyperuricemia leading to bone marrow suppression. People with CKD sufferfrom accelerated atherosclerosis and are more likely to developcardiovascular disease than the general population. Patients afflictedwith CKD and cardiovascular disease tend to have significantly worseprognoses than those suffering only from the latter.

As used herein, “in combination with”, “combinations of”, or “conjointadministration” refers to any form of administration such thatadditional therapies (e.g., second, third, fourth, etc.) are stilleffective in the body (e.g., multiple compounds are simultaneouslyeffective in the patient, which may include synergistic effects of thosecompounds). Effectiveness may not correlate to measurable concentrationof the agent in blood, serum, or plasma. For example, the differenttherapeutic compounds can be administered either in the same formulationor in separate formulations, either concomitantly or sequentially, andon different schedules. Thus, an individual who receives such treatmentcan benefit from a combined effect of different therapies. One or moreALK4:ActRIIB heteromultimer of the disclosure can be administeredconcurrently with, prior to, or subsequent to, one or more otheradditional agents or supportive therapies. In general, each therapeuticagent will be administered at a dose and/or on a time scheduledetermined for that particular agent. The particular combination toemploy in a regimen will take into account compatibility of theantagonist of the present disclosure with the therapy and/or thedesired.

In some embodiments, if a patient has Duchenne Muscular Dystrophy orBecker Muscular Dystrophy, any of the ALK4:ActRIIB heteromultimersdisclosed herein may be administered in combination with one or more of:eteplirsen, a corticosteroid (e.g., deflazacourt), steroids (e.g.,prednisone), a blood pressure and/or heart medication (e.g., angiotensinconverting enzyme inhibitors, beta blockers, and diuretics), ananti-asthmatic (e.g., albuterol), a vitamin/neutrient/antioxidant (e.g.,amino acids, carnitine, coenzyme Q10, creatine, fish oil, green teaextracts, vitamin E), surgery, physical therapy, stem cell therapy, genetherapy, assisted ventilation, diet and/or exercise.

In some embodiments, if a patient has facioscapulohumeral musculardystrophy, any of the ALK4:ActRIIB heteromultimers disclosed herein maybe administered in combination with one or more of: albuterol, speechtherapy, surgery, walking aid, back brace, a T-cell inhibitor (e.g.,truncated histidyl-tRNA synthetase), stem cell therapy, gene therapy,and/or foot support devices.

In some embodiments, if a patient has amytrophic lateral sclerosis, anyof the ALK4:ActRIIB heteromultimers disclosed herein may be administeredin combination with one or more of: riluzole, edaravone, masitinib, anantioxidant, physical therapy, speech therapy, nutritional support,breathing support (e.g., a non-invasive ventilator), stem cell therapy,and/or gene therapy.

In some embodiments, if a patient has sporadic inclusion body myositis,any of the ALK4:ActRIIB heteromultimers disclosed herein may beadministered in combination with one or more of: corticosteroids,prednisone, oxandrolone, methotrexate, mycophenolate mofetil,intravenous immunoglobulin, beta interferon-1a, etanercept, alemtuzumab,follistatin, lithium, bimagrumab, arimoclomol, rapamycin, antioxidants,carnitine, coenzyme Q10, physical therapy, occupational therapy, stemcell therapy, and/or gene therapy.

In some embodiments, if a patient has Alport Syndrome, any of theALK4:ActRIIB heteromultimers disclosed herein may be administered incombination with one or more of: an angiotensin converting enzyme (ACE)inhibitor (e.g., benazepril, cilazapril, enalapril, fosinopril,Lisinopril, perinopril, ramapril and quinapril), an angiotensin receptorblocker (e.g., candesartan, epresartan, irbesartan, losartan,telmisartan and valsartan), a statin (e.g., fluvastatin), anon-dihydropyridine calcium channel blocker (e.g., diltiazem),cyclosporine, and/or aldosterone inhibitors.

In some embodiments, if a patient has sarcopenia, any of theALK4:ActRIIB heteromultimers disclosed herein may be administered incombination with one or more of: urocortin II, hormone replacementtherapy (e.g., testosterone or human growth hormone), creatine, vitaminD, exercise, diet, and/or strength training.

Pulmonary hypertension (PH) has been previously classified as primary(idiopathic) or secondary. Recently, the World Health Organization (WHO)has classified pulmonary hypertension into five groups: Group 1:pulmonary arterial hypertension (PAH); Group 2: pulmonary hypertensionwith left heart disease; Group 3: pulmonary hypertension with lungdisease and/or hypoxemia; Group 4: pulmonary hypertension due to chronicthrombotic and/or embolic disease; and Group 5: miscellaneous conditions(e.g., sarcoidosis, histiocytosis X, lymphangiomatosis and compressionof pulmonary vessels). See, for example, Rubin (2004) Chest 126:7-10.

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity ofpulmonary hypertension (e.g., treating, preventing, or reducing theprogression rate and/or severity of one or more complications ofpulmonary hypertension) comprising administering to a patient in needthereof an effective amount of an ALK4:ActRIIB heteromultimer. In someembodiments, the method relates to pulmonary hypertension patients thathave pulmonary arterial hypertension. In some embodiments, the methodrelates pulmonary hypertension patients that have pulmonary hypertensionwith left heart disease. In some embodiments, the method relates topulmonary hypertension patients that have lung disease and/or hypoxemia.In some embodiments, the method relates to pulmonary hypertensionpatients that have chronic thrombotic and/or embolic disease. In someembodiments, the method relates to pulmonary hypertension patients thathave sarcoidosis, histiocytosis X, or lymphangiomatosis and compressionof pulmonary vessels.

Pulmonary arterial hypertension is a serious, progressive andlife-threatening disease of the pulmonary vasculature, characterized byprofound vasoconstriction and an abnormal proliferation of smooth musclecells in the walls of the pulmonary arteries. Severe constriction of theblood vessels in the lungs leads to very high pulmonary arterialpressures. These high pressures make it difficult for the heart to pumpblood through the lungs to be oxygenated. Patients with PAH suffer fromextreme shortness of breath as the heart struggles to pump against thesehigh pressures. Patients with PAH typically develop significantincreases in pulmonary vascular resistance (PVR) and sustainedelevations in pulmonary artery pressure (PAP), which ultimately lead toright ventricular failure and death. Patients diagnosed with PAH have apoor prognosis and equally compromised quality of life, with a mean lifeexpectancy of 2 to 5 years from the time of diagnosis if untreated.

A variety of factors contribute to the pathogenesis of pulmonaryhypertension including proliferation of pulmonary cells which cancontribute to vascular remodeling (i.e., hyperplasia). For example,pulmonary vascular remodeling occurs primarily by proliferation ofarterial endothelial cells and smooth muscle cells of patients withpulmonary hypertension. Overexpression of various cytokines is believedto promote pulmonary hypertension. Further, it has been found thatpulmonary hypertension may rise from the hyperproliferation of pulmonaryarterial smooth cells and pulmonary endothelial cells. Still further,advanced PAH may be characterized by muscularization of distal pulmonaryarterioles, concentric intimal thickening, and obstruction of thevascular lumen by proliferating endothelial cells. Pietra et al., J. Am.Coll. Cardiol., 43:255-325 (2004).

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity ofpulmonary hypertension (e.g., treating, preventing, or reducing theprogression rate and/or severity of one or more complications ofpulmonary hypertension) comprising administering to a patient in needthereof an effective amount an ALK4:ActRIIB heteromultimer, wherein thepatient has resting pulmonary arterial pressure (PAP) of at least 25 mmHg (e.g., 25, 30, 35, 40, 45, or 50 mm Hg). In some embodiments, themethod relates to patients having a resting PAP of at least 25 mm Hg. Insome embodiments, the method relates to patients having a resting PAP ofat least 30 mm Hg. In some embodiments, the method relates to patientshaving a resting PAP of at least 35 mm Hg. In some embodiments, themethod relates to patients having a resting PAP of at least 40 mm Hg. Insome embodiments, the method relates to patients having a resting PAP ofat least 45 mm Hg. In some embodiments, the method relates to patientshaving a resting PAP of at least 50 mm Hg.

In some embodiments, the disclosure relates to methods of adjusting oneor more hemodynamic parameters in the PH patient toward a more normallevel (e.g., normal as compared to healthy people of similar age andsex), comprising administering to a patient in need thereof an effectiveamount of an ALK4:ActRIIB heteromultimer. In some embodiments, themethod relates to reducing PAP. In some embodiments, the method relatesto reducing the patient's PAP by at least 3 mmHg. In certainembodiments, the method relates to reducing the patient's PAP by atleast 5 mmHg. In certain embodiments, the method relates to reducing thepatient's PAP by at least 7 mmHg. In certain embodiments, the methodrelates to reducing the patient's PAP by at least 10 mmHg. In certainembodiments, the method relates to reducing the patient's PAP by atleast 12 mmHg. In certain embodiments, the method relates to reducingthe patient's PAP by at least 15 mmHg. In certain embodiments, themethod relates to reducing the patient's PAP by at least 20 mmHg. Incertain embodiments, the method relates to reducing the patient's PAP byat least 25 mmHg. In some embodiments, the method relates to reducingpulmonary vascular resistance (PVR). In some embodiments, the methodrelate to increasing pulmonary capillary wedge pressure (PCWP). In someembodiments, the method relate to increasing left ventricularend-diastolic pressure (LVEDP).

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity of one ormore complications of pulmonary hypertension comprising administering toa patient in need thereof an effective amount of an ALK4:ActRIIBheteromultimer. In some embodiments, the method relates to treating,preventing, or reducing the progression rate and/or severity of cellproliferation in the pulmonary artery of a pulmonary hypertensionpatient. In some embodiments, the method relates to treating,preventing, or reducing the progression rate and/or severity of smoothmuscle and/or endothelial cells proliferation in the pulmonary artery ofa pulmonary hypertension patient. In some embodiments, the methodrelates to treating, preventing, or reducing the progression rate and/orseverity of angiogenesis in the pulmonary artery of a pulmonaryhypertension patient. In some embodiments, the method relates toincreasing physical activity of a patient having pulmonary hypertension.In some embodiments, the method relates to treating, preventing, orreducing the progression rate and/or severity of dyspnea in a pulmonaryhypertension patient. In some embodiments, the method relates totreating, preventing, or reducing the progression rate and/or severityof chest pain in a pulmonary hypertension patient. In some embodiments,the method relates to treating, preventing, or reducing the progressionrate and/or severity of fatigue in a pulmonary hypertension patient. Insome embodiments, the method relates to treating, preventing, orreducing the progression rate and/or severity of pulmonary fibrosis in apulmonary hypertension patient. In some embodiments, the method relatesto treating, preventing, or reducing the progression rate and/orseverity of fibrosis in a pulmonary hypertension patient. In someembodiments, the method relates to treating, preventing, or reducing theprogression rate and/or severity of pulmonary vascular remodeling in apulmonary hypertension patient. In some embodiments, the method relatesto treating, preventing, or reducing the progression rate and/orseverity of right ventricular hypertrophy in a pulmonary hypertensionpatient.

In certain aspects, the disclosure relates to methods of increasingexercise capacity in a patient having pulmonary hypertension comprisingadministering to a patient in need thereof an effective amount of anALK4:ActRIIB heteromultimer. Any suitable measure of exercise capacitycan be used. For example, exercise capacity in a 6-minute walk test(6MWT), which measures how far the subject can walk in 6 minutes, i.e.,the 6-minute walk distance (6MWD), is frequently used to assesspulmonary hypertension severity and disease progression. The Borgdyspnea index (BDI) is a numerical scale for assessing perceived dyspnea(breathing discomfort). It measures the degree of breathlessness, forexample, after completion of the 6MWT, where a BDI of 0 indicates nobreathlessness and 10 indicates maximum breathlessness. In someembodiments, the method relates to increasing 6MWD by at least 10 metersin the patient having pulmonary hypertension. In some embodiments, themethod relates to increasing 6MWD by at least 20 meters in the patienthaving pulmonary hypertension. In some embodiments, the method relatesto increasing 6MWD by at least 30 meters in the patient having pulmonaryhypertension. In some embodiments, the method relates to increasing 6MWDby at least 40 meters in the patient having pulmonary hypertension. Insome embodiments, the method relates to increasing 6MWD by at least 50meters in the patient having pulmonary hypertension. In someembodiments, the method relates to increasing 6MWD by at least 60 metersin the patient having pulmonary hypertension. In some embodiments, themethod relates to increasing 6MWD by at least 70 meters in the patienthaving pulmonary hypertension. In some embodiments, the method relatesto increasing 6MWD by at least 80 meters in the patient having pulmonaryhypertension. In some embodiments, the method relates to increasing 6MWDby at least 90 meters in the patient having pulmonary hypertension. Insome embodiments, the method relates to increasing 6MWD by at least 100meters in the patient having pulmonary hypertension. In someembodiments, the method relate to lowering BDI by at least 0.5 indexpoints in the patient having pulmonary hypertension. In someembodiments, the method relate to lowering BDI by at least 1 indexpoints in the patient having pulmonary hypertension. In someembodiments, the method relate to lowering BDI by at least 1.5 indexpoints in the patient having pulmonary hypertension. In someembodiments, the method relate to lowering BDI by at least 2 indexpoints in the patient having pulmonary hypertension. In someembodiments, the method relate to lowering BDI by at least 2.5 indexpoints in the patient having pulmonary hypertension. In someembodiments, the method relate to lowering BDI by at least 3 indexpoints in the patient having pulmonary hypertension. In someembodiments, the method relate to lowering BDI by at least 3.5 indexpoints in the patient having pulmonary hypertension. In someembodiments, the method relate to lowering BDI by at least 4 indexpoints in the patient having pulmonary hypertension. In someembodiments, the method relate to lowering BDI by at least 4.5 indexpoints in the patient having pulmonary hypertension. In someembodiments, the method relate to lowering BDI by at least 5 indexpoints in the patient having pulmonary hypertension. In someembodiments, the method relate to lowering BDI by at least 5.5 indexpoints in the patient having pulmonary hypertension. In someembodiments, the method relate to lowering BDI by at least 6 indexpoints in the patient having pulmonary hypertension. In someembodiments, the method relate to lowering BDI by at least 6.5 indexpoints in the patient having pulmonary hypertension. In someembodiments, the method relate to lowering BDI by at least 7 indexpoints in the patient having pulmonary hypertension. In someembodiments, the method relate to lowering BDI by at least 7.5 indexpoints in the patient having pulmonary hypertension. In someembodiments, the method relate to lowering BDI by at least 8 indexpoints in the patient having pulmonary hypertension. In someembodiments, the method relate to lowering BDI by at least 8.5 indexpoints in the patient having pulmonary hypertension. In someembodiments, the method relate to lowering BDI by at least 9 indexpoints in the patient having pulmonary hypertension. In someembodiments, the method relate to lowering BDI by at least 9.5 indexpoints in the patient having pulmonary hypertension. In someembodiments, the method relate to lowering BDI by at least 3 indexpoints in the patient having pulmonary hypertension. In someembodiments, the method relate to lowering BDI by 10 index points in thepatient having pulmonary hypertension.

Pulmonary hypertension at baseline can be mild, moderate or severe, asmeasured for example by World Health Organization (WHO) functionalclass, which is a measure of disease severity in patients with pulmonaryhypertension. The WHO functional classification is an adaptation of theNew York Heart Association (NYHA) system and is routinely used toqualitatively assess activity tolerance, for example in monitoringdisease progression and response to treatment (Rubin (2004) Chest126:7-10). Four functional classes are recognized in the WHO system:Class I: pulmonary hypertension without resulting limitation of physicalactivity; ordinary physical activity does not cause undue dyspnea orfatigue, chest pain or near syncope; Class II: pulmonary hypertensionresulting in slight limitation of physical activity; patient comfortableat rest; ordinary physical activity causes undue dyspnea or fatigue,chest pain or near syncope; Class III: pulmonary hypertension resultingin marked limitation of physical activity; patient comfortable at rest;less than ordinary activity causes undue dyspnea or fatigue, chest painor near syncope; Class IV: pulmonary hypertension resulting in inabilityto carry out any physical activity without symptoms; patient manifestssigns of right-heart failure; dyspnea and/or fatigue may be present evenat rest; discomfort is increased by any physical activity.

In certain aspects, the disclosure relates to methods of treating,preventing, or reducing the progression rate and/or severity ofpulmonary hypertension (e.g., treating, preventing, or reducing theprogression rate and/or severity of one or more complications ofpulmonary hypertension) comprising administering to a patient in needthereof an effective amount of an ALK4:ActRIIB heteromultimer, whereinthe patient has Class I, Class II, Class III, or Class IV pulmonaryhypertension as recognized by the WHO. In some embodiments, the methodrelates to a patient that has Class I pulmonary hypertension asrecognized by the WHO. In some embodiments, the method relates to apatient that has Class II pulmonary hypertension as recognized by theWHO. In some embodiments, the method relates to preventing or delayingpatient progression from Class I pulmonary hypertension to Class IIpulmonary hypertension as recognized by the WHO. In some embodiments,the method relates to promoting or increasing patient regression fromClass II pulmonary hypertension to Class I pulmonary hypertension asrecognized by the WHO. In some embodiments, the method relates to apatient that has Class III pulmonary hypertension as recognized by theWHO. In some embodiments, the method relates to preventing or delayingpatient progression from Class II pulmonary hypertension to Class IIIpulmonary hypertension as recognized by the WHO. In some embodiments,the method relates to promoting or increasing patient regression fromClass III pulmonary hypertension to Class II pulmonary hypertension asrecognized by the WHO. In some embodiments, the method relates topromoting or increasing patient regression from Class III pulmonaryhypertension to Class I pulmonary hypertension as recognized by the WHO.In some embodiments, the method relates to a patient that has Class IVpulmonary hypertension as recognized by the WHO. In some embodiments,the method relates to preventing or delaying patient progression fromClass III pulmonary hypertension to Class IV pulmonary hypertension asrecognized by the WHO. In some embodiments, the method relates topromoting or increasing patient regression from Class IV pulmonaryhypertension to Class III pulmonary hypertension as recognized by theWHO. In some embodiments, the method relates to promoting or increasingpatient regression from Class IV pulmonary hypertension to Class IIpulmonary hypertension as recognized by the WHO. In some embodiments,the method relates to promoting or increasing patient regression fromClass IV pulmonary hypertension to Class I pulmonary hypertension asrecognized by the WHO.

There is no known cure for pulmonary hypertension; current methods oftreatment focus on prolonging patient lifespan and enhancing patientquality of life. Current methods of treatment of pulmonary hypertensioninclude administration of: vasodilators such as prostacyclin,epoprostenol, and sildenafil; endothelin receptor antagonists such asbosentan; calcium channel blockers such as amlodipine, diltiazem, andnifedipine; anticoagulants such as warfarin; and diuretics. Treatment ofpulmonary hypertension has also been carried out using oxygen therapy,atrial septostomy, pulmonary thromboendarterectomy, and lung and/orheart transplantation. Each of these methods, however, suffers from oneor multiple drawbacks which may include lack of effectiveness, seriousside effects, low patient compliance, and high cost. In certain aspects,the method relate to treating, preventing, or reducing the progressionrate and/or severity of pulmonary hypertension (e.g., treating,preventing, or reducing the progression rate and/or severity of one ormore complications of pulmonary hypertension) comprising administeringto a patient in need thereof an effective amount of an ALK4:ActRIIBheteromultimer in combination (e.g., administered at the same time ordifferent times, but generally in such a manner as to achieveoverlapping pharmacological/physiological effects) with one or moreadditional active agents and/or supportive therapies for treatingpulmonary hypertension (e.g., vasodilators such as prostacyclin,epoprostenol, and sildenafil; endothelin receptor antagonists such asbosentan; calcium channel blockers such as amlodipine, diltiazem, andnifedipine; anticoagulants such as warfarin; diuretics; oxygen therapy;atrial septostomy; pulmonary thromboendarterectomy: and lung and/orheart transplantation); BMP9 polypeptides; BMP10 polypeptides;bardoxolone methyl or a derivative thereof oleanolic acid or derivativethereof.

5. Pharmaceutical Compositions

In certain aspects, ALK4:ActRIIB heteromultimers of the presentdisclosure can be administered alone or as a component of apharmaceutical formulation (also referred to as a therapeuticcomposition or pharmaceutical composition). A pharmaceutical formationrefers to a preparation which is in such form as to permit thebiological activity of an active ingredient (e.g., an agent of thepresent disclosure) contained therein to be effective and which containsno additional components which are unacceptably toxic to a subject towhich the formulation would be administered. The subject compounds maybe formulated for administration in any convenient way for use in humanor veterinary medicine. For example, one or more agents of the presentdisclosure may be formulated with a pharmaceutically acceptable carrier.A pharmaceutically acceptable carrier refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isgenerally nontoxic to a subject. A pharmaceutically acceptable carrierincludes, but is not limited to, a buffer, excipient, stabilizer, and/orpreservative. In general, pharmaceutical formulations for use in thepresent disclosure are in a pyrogen-free, physiologically-acceptableform when administered to a subject. Therapeutically useful agents otherthan those described herein, which may optionally be included in theformulation as described above, may be administered in combination withthe subject agents in the methods of the present disclosure.

In certain embodiments, compositions will be administered parenterally[e.g., by intravenous (I.V.) injection, intraarterial injection,intraosseous injection, intramuscular injection, intrathecal injection,subcutaneous injection, or intradermal injection].

Pharmaceutical compositions suitable for parenteral administration maycomprise one or more agents of the disclosure in combination with one ormore pharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use. Injectable solutions or dispersions maycontain antioxidants, buffers, bacteriostats, suspending agents,thickening agents, or solutes which render the formulation isotonic withthe blood of the intended recipient. Examples of suitable aqueous andnonaqueous carriers which may be employed in the pharmaceuticalformulations of the present disclosure include water, ethanol, polyols(e.g., glycerol, propylene glycol, polyethylene glycol, etc.), vegetableoils (e.g., olive oil), injectable organic esters (e.g., ethyl oleate),and suitable mixtures thereof. Proper fluidity can be maintained, forexample, by the use of coating materials (e.g., lecithin), by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

In some embodiments, a therapeutic method of the present disclosureincludes administering the pharmaceutical composition systemically, orlocally, from an implant or device. Further, the pharmaceuticalcomposition may be encapsulated or injected in a form for delivery to atarget tissue site (e.g., bone marrow or muscle). In certainembodiments, compositions of the present disclosure may include a matrixcapable of delivering one or more of the agents of the presentdisclosure to a target tissue site (e.g., bone marrow or muscle),providing a structure for the developing tissue and optimally capable ofbeing resorbed into the body. For example, the matrix may provide slowrelease of one or more agents of the present disclosure. Such matricesmay be formed of materials presently in use for other implanted medicalapplications.

The choice of matrix material may be based on one or more of:biocompatibility, biodegradability, mechanical properties, cosmeticappearance, and interface properties. The particular application of thesubject compositions will define the appropriate formulation. Potentialmatrices for the compositions may be biodegradable and chemicallydefined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylacticacid, and polyanhydrides. Other potential materials are biodegradableand biologically well-defined including, for example, bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenon-biodegradable and chemically defined including, for example,sintered hydroxyapatite, bioglass, aluminates, or other ceramics.Matrices may be comprised of combinations of any of the above mentionedtypes of material including, for example, polylactic acid andhydroxyapatite or collagen and tricalciumphosphate. The bioceramics maybe altered in composition (e.g., calcium-aluminate-phosphate) andprocessing to alter one or more of pore size, particle size, particleshape, and biodegradability.

In certain embodiments, pharmaceutical compositions of presentdisclosure can be administered topically. “Topical application” or“topically” means contact of the pharmaceutical composition with bodysurfaces including, for example, the skin, wound sites, and mucousmembranes. The topical pharmaceutical compositions can have variousapplication forms and typically comprises a drug-containing layer, whichis adapted to be placed near to or in direct contact with the tissueupon topically administering the composition. Pharmaceuticalcompositions suitable for topical administration may comprise one ormore one or more ALK4:ActRIIB heteromultimers of the disclosure incombination formulated as a liquid, a gel, a cream, a lotion, anointment, a foam, a paste, a putty, a semi-solid, or a solid.Compositions in the liquid, gel, cream, lotion, ointment, foam, paste,or putty form can be applied by spreading, spraying, smearing, dabbingor rolling the composition on the target tissue. The compositions alsomay be impregnated into sterile dressings, transdermal patches,plasters, and bandages. Compositions of the putty, semi-solid or solidforms may be deformable. They may be elastic or non-elastic (e.g.,flexible or rigid). In certain aspects, the composition forms part of acomposite and can include fibers, particulates, or multiple layers withthe same or different compositions.

Topical compositions in the liquid form may include pharmaceuticallyacceptable solutions, emulsions, microemulsions, and suspensions. Inaddition to the active ingredient(s), the liquid dosage form may containan inert diluent commonly used in the art including, for example, wateror other solvent, a solubilizing agent and/or emulsifier [e.g., ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, or 1,3-butylene glycol, anoil (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesameoil), glycerol, tetrahydrofuryl alcohol, a polyethylene glycol, a fattyacid ester of sorbitan, and mixtures thereof].

Topical gel, cream, lotion, ointment, semi-solid or solid compositionsmay include one or more thickening agents, such as a polysaccharide,synthetic polymer or protein-based polymer. In one embodiment of theinvention, the gelling agent herein is one that is suitably nontoxic andgives the desired viscosity. The thickening agents may include polymers,copolymers, and monomers of: vinylpyrrolidones, methacrylamides,acrylamides N-vinylimidazoles, carboxy vinyls, vinyl esters, vinylethers, silicones, polyethyleneoxides, polyethyleneglycols,vinylalcohols, sodium acrylates, acrylates, maleic acids,NN-dimethylacrylamides, diacetone acrylamides, acrylamides, acryloylmorpholine, pluronic, collagens, polyacrylamides, polyacrylates,polyvinyl alcohols, polyvinylenes, polyvinyl silicates, polyacrylatessubstituted with a sugar (e.g., sucrose, glucose, glucosamines,galactose, trehalose, mannose, or lactose), acylamidopropane sulfonicacids, tetramethoxyorthosilicates, methyltrimethoxyorthosilicates,tetraalkoxyorthosilicates, trialkoxyorthosilicates, glycols, propyleneglycol, glycerine, polysaccharides, alginates, dextrans, cyclodextrin,celluloses, modified celluloses, oxidized celluloses, chitosans,chitins, guars, carrageenans, hyaluronic acids, inulin, starches,modified starches, agarose, methylcelluloses, plant gums, hylaronans,hydrogels, gelatins, glycosaminoglycans, carboxymethyl celluloses,hydroxyethyl celluloses, hydroxy propyl methyl celluloses, pectins,low-methoxy pectins, cross-linked dextrans, starch-acrylonitrile graftcopolymers, starch sodium polyacrylate, hydroxyethyl methacrylates,hydroxyl ethyl acrylates, polyvinylene, polyethylvinylethers, polymethylmethacrylates, polystyrenes, polyurethanes, polyalkanoates, polylacticacids, polylactates, poly(3-hydroxybutyrate), sulfonated hydrogels, AMPS(2-acrylamido-2-methyl-1-propanesulfonic acid), SEM(sulfoethylmethacrylate), SPM (sulfopropyl methacrylate), SPA(sulfopropyl acrylate),N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine,methacryllic acid amidopropyl-dimethyl ammonium sulfobetaine, SPI(itaconic acid-bis(1-propyl sulfonizacid-3) ester di-potassium salt),itaconic acids, AMBC (3-acrylamido-3-methylbutanoic acid),beta-carboxyethyl acrylate (acrylic acid dimers), and maleicanhydride-methylvinyl ether polymers, derivatives thereof, saltsthereof, acids thereof, and combinations thereof. In certainembodiments, pharmaceutical compositions of present disclosure can beadministered orally, for example, in the form of capsules, cachets,pills, tablets, lozenges (using a flavored basis such as sucrose andacacia or tragacanth), powders, granules, a solution or a suspension inan aqueous or non-aqueous liquid, an oil-in-water or water-in-oil liquidemulsion, or an elixir or syrup, or pastille (using an inert base, suchas gelatin and glycerin, or sucrose and acacia), and/or a mouth wash,each containing a predetermined amount of a compound of the presentdisclosure and optionally one or more other active ingredients. Acompound of the present disclosure and optionally one or more otheractive ingredients may also be administered as a bolus, electuary, orpaste.

In solid dosage forms for oral administration (e.g., capsules, tablets,pills, dragees, powders, and granules), one or more compounds of thepresent disclosure may be mixed with one or more pharmaceuticallyacceptable carriers including, for example, sodium citrate, dicalciumphosphate, a filler or extender (e.g., a starch, lactose, sucrose,glucose, mannitol, and silicic acid), a binder (e.g.carboxymethylcellulose, an alginate, gelatin, polyvinyl pyrrolidone,sucrose, and acacia), a humectant (e.g., glycerol), a disintegratingagent (e.g., agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, a silicate, and sodium carbonate), a solution retardingagent (e.g. paraffin), an absorption accelerator (e.g. a quaternaryammonium compound), a wetting agent (e.g., cetyl alcohol and glycerolmonostearate), an absorbent (e.g., kaolin and bentonite clay), alubricant (e.g., a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate), a coloring agent, andmixtures thereof. In the case of capsules, tablets, and pills, thepharmaceutical formulation (composition) may also comprise a bufferingagent. Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using one or moreexcipients including, e.g., lactose or a milk sugar as well as a highmolecular-weight polyethylene glycol.

Liquid dosage forms for oral administration of the pharmaceuticalcomposition may include pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and elixirs. In additionto the active ingredient(s), the liquid dosage form may contain an inertdiluent commonly used in the art including, for example, water or othersolvent, a solubilizing agent and/or emulsifier [e.g., ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, or 1,3-butylene glycol, an oil (e.g.,cottonseed, groundnut, corn, germ, olive, castor, and sesame oil),glycerol, tetrahydrofuryl alcohol, a polyethylene glycol, a fatty acidester of sorbitan, and mixtures thereof]. Besides inert diluents, theoral formulation can also include an adjuvant including, for example, awetting agent, an emulsifying and suspending agent, a sweetening agent,a flavoring agent, a coloring agent, a perfuming agent, a preservativeagent, and combinations thereof.

Suspensions, in addition to the active compounds, may contain suspendingagents including, for example, an ethoxylated isostearyl alcohol,polyoxyethylene sorbitol, a sorbitan ester, microcrystalline cellulose,aluminum metahydroxide, bentonite, agar-agar, tragacanth, andcombinations thereof.

Prevention of the action and/or growth of microorganisms may be ensuredby the inclusion of various antibacterial and antifungal agentsincluding, for example, paraben, chlorobutanol, and phenol sorbic acid.

In certain embodiments, it may be desirable to include an isotonic agentincluding, for example, a sugar or sodium chloride into thecompositions. In addition, prolonged absorption of an injectablepharmaceutical form may be brought about by the inclusion of an agentthat delay absorption including, for example, aluminum monostearate andgelatin.

It is understood that the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the one or more of the agents of the present disclosure. In the caseof a ALK4:ActRIIB heteromultimer that promotes red blood cell formation,various factors may include, but are not limited to, the patient's redblood cell count, hemoglobin level, the desired target red blood cellcount, the patient's age, the patient's sex, the patient's diet, theseverity of any disease that may be contributing to a depressed redblood cell level, the time of administration, and other clinicalfactors. The addition of other known active agents to the finalcomposition may also affect the dosage. Progress can be monitored byperiodic assessment of one or more of red blood cell levels, hemoglobinlevels, reticulocyte levels, and other indicators of the hematopoieticprocess.

In certain embodiments, the present disclosure also provides genetherapy for the in vivo production of one or more of the agents of thepresent disclosure. Such therapy would achieve its therapeutic effect byintroduction of the agent sequences into cells or tissues having one ormore of the disorders as listed above. Delivery of the agent sequencescan be achieved, for example, by using a recombinant expression vectorsuch as a chimeric virus or a colloidal dispersion system. Preferredtherapeutic delivery of one or more of agent sequences of the disclosureis the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or an RNA virus(e.g., a retrovirus). The retroviral vector may be a derivative of amurine or avian retrovirus. Examples of retroviral vectors in which asingle foreign gene can be inserted include, but are not limited to:Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus(HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus(RSV). A number of additional retroviral vectors can incorporatemultiple genes. All of these vectors can transfer or incorporate a genefor a selectable marker so that transduced cells can be identified andgenerated. Retroviral vectors can be made target-specific by attaching,for example, a sugar, a glycolipid, or a protein. Preferred targeting isaccomplished by using an antibody. Those of skill in the art willrecognize that specific polynucleotide sequences can be inserted intothe retroviral genome or attached to a viral envelope to allow targetspecific delivery of the retroviral vector containing one or more of theagents of the present disclosure.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes (gag, pol, and env),by conventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for one or more of the agents of thepresent disclosure is a colloidal dispersion system. Colloidaldispersion systems include, for example, macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Incertain embodiments, the preferred colloidal system of this disclosureis a liposome. Liposomes are artificial membrane vesicles which areuseful as delivery vehicles in vitro and in vivo. RNA, DNA, and intactvirions can be encapsulated within the aqueous interior and be deliveredto cells in a biologically active form [Fraley, et al. (1981) TrendsBiochem. Sci., 6:77]. Methods for efficient gene transfer using aliposome vehicle are known in the art [Mannino, et al. (1988)Biotechniques, 6:682, 1988].

The composition of the liposome is usually a combination ofphospholipids, which may include a steroid (e.g. cholesterol). Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations. Other phospholipids or other lipidsmay also be used including, for example a phosphatidyl compound (e.g.,phosphatidylglycerol, phosphatidylcholine, phosphatidylserine,phosphatidylethanolamine, a sphingolipid, a cerebroside, and aganglioside), egg phosphatidylcholine, dipalmitoylphosphatidylcholine,and distearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present invention, and are not intended to limit theinvention.

Example 1. Generation of an ALK4:ActRIIB Heterodimer

Soluble ALK4-Fc:ActRIIB-Fc heteromeric complexes comprising theextracellular domains of human ActRIIB and human ALK4, which are eachseparately fused to an Fc domain with a linker positioned between theextracellular domain and the Fc domain, were constructed. The individualconstructs are referred to as ActRIIB-Fc fusion polypeptide and ALK4-Fcfusion polypeptide, respectively, and the sequences for each areprovided below.

A methodology for promoting formation of ALK4-Fc:ActRIIB-Fc heteromericcomplexes, as opposed to ActRIIB-Fc or ALK4-Fc homodimeric complexes, isto introduce alterations in the amino acid sequence of the Fc domains toguide the formation of asymmetric heteromeric complexes. Many differentapproaches to making asymmetric interaction pairs using Fc domains aredescribed in this disclosure.

In one approach, illustrated in the ActRIIB-Fc and ALK4-Fc polypeptidesequences of SEQ ID NOs: 39-41 and 42-44, respectively, one Fc domain isaltered to introduce cationic amino acids at the interaction face, whilethe other Fc domain is altered to introduce anionic amino acids at theinteraction face. ActRIIB-Fc fusion polypeptide and ALK4-Fc fusionpolypeptide each employ the tissue plasminogen activator (TPA) leader:

(SEQ ID NO: 38) MDAMKRGLCCVLLLCGAVFVSP.

The ActRIIB-Fc polypeptide sequence (SEQ ID NO: 39) is shown below:

(SEQ ID NO: 39) 1 MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS51 GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDFNC YDRQECVATE 101 ENPQVYFCCCEGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC 151 PAPELLGGPS VFLFPPKPKDTLMISRTPEV TCVVVDVSHE DPEVKFNWYV 201 DGVEVHNAKT KPREEQYNST YRVVSVLTVLHQDWLNGKEY KCKVSNKALP 251 APIEKTISKA KGQPREPQVY TLPPSRKEMT KNQVSLTCLVKGFYPSDIAV 301 EWESNGQPEN NYKTTPPVLK SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH351 EALHNHYTQK SLSLSPGK

The leader (signal) sequence and linker are underlined. To promoteformation of ALK4-Fc:ActRIIB-Fc heterodimer rather than either of thepossible homodimeric complexes, two amino acid substitutions (replacingacidic amino acids with lysine) can be introduced into the Fc domain ofthe ActRIIB fusion protein as indicated by double underline above. Theamino acid sequence of SEQ ID NO: 39 may optionally be provided withlysine (K) removed from the C-terminus.

This ActRIIB-Fc fusion protein is encoded by the following nucleic acidsequence (SEQ ID NO: 40):

(SEQ ID NO: 40) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC51 AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT GAGACACGGG 101 AGTGCATCTACTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGAGC 151 GGCCTGGAGC GCTGCGAAGGCGAGCAGGAC AAGCGGCTGC ACTGCTACGC 201 CTCCTGGCGC AACAGCTCTG GCACCATCGAGCTCGTGAAG AAGGGCTGCT 251 GGCTAGATGA CTTCAACTGC TACGATAGGC AGGAGTGTGTGGCCACTGAG 301 GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT TCTGCAACGA351 GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC 401CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG CCCACCGTGC 451 CCAGCACCTGAACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA 501 ACCCAAGGAC ACCCTCATGATCTCCCGGAC CCCTGAGGTC ACATGCGTGG 551 TGGTGGACGT GAGCCACGAA GACCCTGAGGTCAAGTTCAA CTGGTACGTG 601 GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGGAGGAGCAGTA 651 CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT701 GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA 751GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC 801 ACAGGTGTACACCCTGCCCC CATCCCGGAA GGAGATGACC AAGAACCAGG 851 TCAGCCTGAC CTGCCTGGTCAAAGGCTTCT ATCCCAGCGA CATCGCCGTG 901 GAGTGGGAGA GCAATGGGCA GCCGGAGAACAACTACAAGA CCACGCCTCC 951 CGTGCTGAAG TCCGACGGCT CCTTCTTCCT CTATAGCAAGCTCACCGTGG 1001 ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT1051 GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG 1101 TAAA

The mature ActRIIB-Fc fusion polypeptide (SEQ ID NO: 41) is as follows,and may optionally be provided with lysine (K) removed from theC-terminus.

(SEQ ID NO: 41) 1 GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT51 IELVKKGCWL DDFNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA 101 GGPEVTYEPPPTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS 151 RTPEVTCVVV DVSHEDPEVKFNWYVDGVEV HNAKTKPREE QYNSTYRVVS 201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEKTISKAKGQPR EPQVYTLPPS 251 RKEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTTPPVLKSDGSF 301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

The complementary form of ALK4-Fc fusion polypeptide (SEQ ID NO: 42) isas follows:

(SEQ ID NO: 42) 1 MDAMKRGLCC VLLLCGAVFV SPGASGPRGV QALLCACTSC LQANYTCETD51 GACMVSIFNL DGMEHHVRTC IPKVELVPAG KPFYCLSSED LRNTHCCYTD 101 YCNRIDLRVPSGHLKEPEHP SMWGPVETGG GTHTCPPCPA PELLGGPSVF 151 LFPPKPKDTL MISRTPEVTCVVVDVSHEDP EVKFNWYVDG VEVHNAKTKP 201 REEQYNSTYR VVSVLTVLHQ DWLNGKEYKCKVSNKALPAP IEKTISKAKG 251 QPREPQVYTL PPSREEMTKN QVSLTCLVKG FYPSDIAVEWESNGQPENNY 301 DTTPPVLDSD GSFFLYSDLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL351 SLSPG

The leader sequence and linker are underlined. To guide heterodimerformation with the ActRIIB-Fc fusion polypeptide of SEQ ID NOs: 39 and41 above, two amino acid substitutions (replacing lysines with asparticacids) can be introduced into the Fc domain of the ALK4-Fc fusionpolypeptide as indicated by double underline above. The amino acidsequence of SEQ ID NO: 42 may optionally be provided with lysine (K)added at the C-terminus.

This ALK4-Fc fusion protein is encoded by the following nucleic acid(SEQ ID NO: 43):

(SEQ ID NO: 43) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC51 AGTCTTCGTT TCGCCCGGCG CCTCCGGGCC CCGGGGGGTC CAGGCTCTGC 101 TGTGTGCGTGCACCAGCTGC CTCCAGGCCA ACTACACGTG TGAGACAGAT 151 GGGGCCTGCA TGGTTTCCATTTTCAATCTG GATGGGATGG AGCACCATGT 201 GCGCACCTGC ATCCCCAAAG TGGAGCTGGTCCCTGCCGGG AAGCCCTTCT 251 ACTGCCTGAG CTCGGAGGAC CTGCGCAACA CCCACTGCTGCTACACTGAC 301 TACTGCAACA GGATCGACTT GAGGGTGCCC AGTGGTCACC TCAAGGAGCC351 TGAGCACCCG TCCATGTGGG GCCCGGTGGA GACCGGTGGT GGAACTCACA 401CATGCCCACC GTGCCCAGCA CCTGAACTCC TGGGGGGACC GTCAGTCTTC 451 CTCTTCCCCCCAAAACCCAA GGACACCCTC ATGATCTCCC GGACCCCTGA 501 GGTCACATGC GTGGTGGTGGACGTGAGCCA CGAAGACCCT GAGGTCAAGT 551 TCAACTGGTA CGTGGACGGC GTGGAGGTGCATAATGCCAA GACAAAGCCG 601 CGGGAGGAGC AGTACAACAG CACGTACCGT GTGGTCAGCGTCCTCACCGT 651 CCTGCACCAG GACTGGCTGA ATGGCAAGGA GTACAAGTGC AAGGTCTCCA701 ACAAAGCCCT CCCAGCCCCC ATCGAGAAAA CCATCTCCAA AGCCAAAGGG 751CAGCCCCGAG AACCACAGGT GTACACCCTG CCCCCATCCC GGGAGGAGAT 801 GACCAAGAACCAGGTCAGCC TGACCTGCCT GGTCAAAGGC TTCTATCCCA 851 GCGACATCGC CGTGGAGTGGGAGAGCAATG GGCAGCCGGA GAACAACTAC 901 GACACCACGC CTCCCGTGCT GGACTCCGACGGCTCCTTCT TCCTCTATAG 951 CGACCTCACC GTGGACAAGA GCAGGTGGCA GCAGGGGAACGTCTTCTCAT 1001 GCTCCGTGAT GCATGAGGCT CTGCACAACC ACTACACGCA GAAGAGCCTC1051 TCCCTGTCTC CGGGT

The mature ALK4-Fc fusion protein sequence (SEQ ID NO: 44) is as followsand may optionally be provided with lysine (K) added at the C-terminus.

(SEQ ID NO: 44) 1 SGPRGVQALL CACTSCLQAN YTCETDGACM VSIFNLDGME HHVRTCIPKV51 ELVPAGKPFY CLSSEDLRNT HCCYTDYCNR IDLRVPSGHL KEPEHPSMWG 101 PVETGGGTHTCPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD 151 VSHEDPEVKF NWYVDGVEVHNAKTKPREEQ YNSTYRVVSV LTVLHQDWLN 201 GKEYKCKVSN KALPAPIEKT ISKAKGQPREPQVYTLPPSR EEMTKNQVSL 251 TCLVKGFYPS DIAVEWESNG QPENNYDTTP PVLDSDGSFFLYSDLTVDKS 301 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP G

The ActRIIB-Fc and ALK4-Fc proteins of SEQ ID NO: 41 and SEQ ID NO: 44,respectively, may be co-expressed and purified from a CHO cell line, togive rise to a heteromeric complex comprising ALK4-Fc:ActRIIB-Fc.

In another approach to promote the formation of heteromultimer complexesusing asymmetric Fc fusion proteins the Fc domains are altered tointroduce complementary hydrophobic interactions and an additionalintermolecular disulfide bond as illustrated in the ActRIIB-Fc andALK4-Fc polypeptide sequences of SEQ ID NOs: 45-46 and 47-48,respectively. The ActRIIB-Fc fusion polypeptide and ALK4-Fc fusionpolypeptide each employ the tissue plasminogen activator (TPA) leader:MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 38).

The ActRIIB-Fc polypeptide sequence (SEQ ID NO: 45) is shown below:

(SEQ ID NO: 45) 1 MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS51 GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDFNC YDRQECVATE 101 ENPQVYFCCCEGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC 151 PAPELLGGPS VFLFPPKPKDTLMISRTPEV TCVVVDVSHE DPEVKFNWYV 201 DGVEVHNAKT KPREEQYNST YRVVSVLTVLHQDWLNGKEY KCKVSNKALP 251 APIEKTISKA KGQPREPQVY TLPPCREEMT KNQVSLWCLVKGFYPSDIAV 301 EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH351 EALHNHYTQK SLSLSPGK

The leader (signal) sequence and linker are underlined. To promoteformation of the ALK4-Fc:ActRIIB-Fc heterodimer rather than either ofthe possible homodimeric complexes, two amino acid substitutions(replacing a serine with a cysteine and a threonine with a trytophan)can be introduced into the Fc domain of the fusion protein as indicatedby double underline above. The amino acid sequence of SEQ ID NO: 45 mayoptionally be provided with lysine (K) removed from the C-terminus.

The mature ActRIIB-Fc fusion polypeptide is as follows:

(SEQ ID NO: 46) 1 GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT51 IELVKKGCWL DDFNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA 101 GGPEVTYEPPPTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS 151 RTPEVTCVVV DVSHEDPEVKFNWYVDGVEV HNAKTKPREE QYNSTYRVVS 201 VLTVLHQDWL NGKEYKCKVS NKALPAPIEKTISKAKGQPR EPQVYTLPPC 251 REEMTKNQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTTPPVLDSDGSF 301 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK

The complementary form of ALK4-Fc fusion polypeptide (SEQ ID NO: 47) isas follows and may optionally be provided with lysine (K) removed fromthe C-terminus.

(SEQ ID NO: 47) 1 MDAMKRGLCC VLLLCGAVFV SPGASGPRGV QALLCACTSC LQANYTCETD51 GACMVSIFNL DGMEHHVRTC IPKVELVPAG KPFYCLSSED LRNTHCCYTD 101 YCNRIDLRVPSGHLKEPEHP SMWGPVETGG GTHTCPPCPA PELLGGPSVF 151 LFPPKPKDTL MISRTPEVTCVVVDVSHEDP EVKFNWYVDG VEVHNAKTKP 201 REEQYNSTYR VVSVLTVLHQ DWLNGKEYKCKVSNKALPAP IEKTISKAKG 251 QPREPQVCTL PPSREEMTKN QVSLSCAVKG FYPSDIAVEWESNGQPENNY 301 KTTPPVLDSD GSFFLVSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL351 SLSPGK

The leader sequence and the linker are underlined. To guide heterodimerformation with the ActRIIB-Fc fusion polypeptide of SEQ ID NOs: 45 and46 above, four amino acid substitutions can be introduced into the Fcdomain of the ALK4 fusion polypeptide as indicated by double underlineabove. The amino acid sequence of SEQ ID NO: 47 may optionally beprovided with lysine (K) removed from the C-terminus.

The mature ALK4-Fc fusion protein sequence is as follows and mayoptionally be provided with lysine (K) removed from the C-terminus.

(SEQ ID NO: 48) 1 SGPRGVQALL CACTSCLQAN YTCETDGACM VSIFNLDGME HHVRTCIPKV51 ELVPAGKPFY CLSSEDLRNT HCCYTDYCNR IDLRVPSGHL KEPEHPSMWG 101PVETGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD 151VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN 201GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVCTLPPSR EEMTKNQVSL 251SCAVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LVSKLTVDKS 301RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK

ActRIIB-Fc and ALK4-Fc proteins of SEQ ID NO: 46 and SEQ ID NO: 48,respectively, may be co-expressed and purified from a CHO cell line, togive rise to a heteromeric complex comprising ALK4-Fc:ActRIIB-Fc.

Purification of various ALK4-Fc:ActRIIB-Fc complexes could be achievedby a series of column chromatography steps, including, for example,three or more of the following, in any order: protein A chromatography,Q sepharose chromatography, phenylsepharose chromatography, sizeexclusion chromatography, cation exchange chromatography, andepitope-based affinity chromatography (e.g., with an antibody orfunctionally equivalent ligand directed against an epitope on ALK4 orActRIIB). The purification could be completed with viral filtration andbuffer exchange.

In another approach to promote the formation of heteromultimer complexesusing asymmetric Fc fusion proteins, the Fc domains are altered tointroduce complementary hydrophobic interactions, an additionalintermolecular disulfide bond, and electrostatic differences between thetwo Fc domains for facilitating purification based on net molecularcharge, as illustrated in the ActRIIB-Fc and ALK4-Fc polypeptidesequences of SEQ ID NOs: 70-73 and 74-77, respectively. The ActRIIB-Fcfusion polypeptide and ALK4-Fc fusion polypeptide each employ the tissueplasminogen activator (TPA) leader: MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO:38).

The ActRIIB-Fc polypeptide sequence (SEQ ID NO: 70) is shown below:

(SEQ ID NO: 70) 1 MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS51 GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDFNC YDRQECVATE 101ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC 151PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV 201DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP 251APIEKTISKA KGQPREPQVY TLPPCREEMT ENQVSLWCLV KGFYPSDIAV 301EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH 351 EALHNHYTQDSLSLSPG

The leader sequence and linker are underlined. To promote formation ofthe ALK4-Fc:ActRIIB-Fc heterodimer rather than either of the possiblehomodimeric complexes, two amino acid substitutions (replacing a serinewith a cysteine and a threonine with a trytophan) can be introduced intothe Fc domain of the fusion protein as indicated by double underlineabove. To facilitate purification of the ALK4-Fc:ActRIIB-Fc heterodimer,two amino acid substitutions (replacing lysines with acidic amino acids)can also be introduced into the Fc domain of the fusion protein asindicated by double underline above. The amino acid sequence of SEQ IDNO: 70 may optionally be provided with a lysine added at the C-terminus.

This ActRIIB-Fc fusion protein is encoded by the following nucleic acid(SEQ ID NO: 71):

(SEQ ID NO: 71) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC51 AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT GAGACACGGG 101AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGAGC 151GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC ACTGCTACGC 201CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT 251GGCTAGATGA CTTCAACTGC TACGATAGGC AGGAGTGTGT GGCCACTGAG 301GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT TCTGCAACGA 351GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC 401CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG CCCACCGTGC 451CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA 501ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG 551TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG 601GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA 651CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT 701GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA 751GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC 801ACAGGTGTAC ACCCTGCCCC CATGCCGGGA GGAGATGACC GAGAACCAGG 851TCAGCCTGTG GTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG 901GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC 951CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTATAGCAAG CTCACCGTGG 1001ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT 1051GAGGCTCTGC ACAACCACTA CACGCAGGAC AGCCTCTCCC TGTCTCCGGG 1101 T

The mature ActRIIB-Fc fusion polypeptide is as follows (SEQ ID NO: 72)and may optionally be provided with a lysine added to the C-terminus.

(SEQ ID NO: 72) 1 GRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT51 IELVKKGCWL DDFNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA 101GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS 151RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS 201VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPC 251REEMTENQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF 301FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQDSLSLS PG

This ActRIIB-Fc fusion polypeptide is encoded by the following nucleicacid (SEQ ID NO: 73):

(SEQ ID NO: 73) 1 GGGCGTGGGG AGGCTGAGAC ACGGGAGTGC ATCTACTACA ACGCCAACTG51 GGAGCTGGAG CGCACCAACC AGAGCGGCCT GGAGCGCTGC GAAGGCGAGC 101AGGACAAGCG GCTGCACTGC TACGCCTCCT GGCGCAACAG CTCTGGCACC 151ATCGAGCTCG TGAAGAAGGG CTGCTGGCTA GATGACTTCA ACTGCTACGA 201TAGGCAGGAG TGTGTGGCCA CTGAGGAGAA CCCCCAGGTG TACTTCTGCT 251GCTGTGAAGG CAACTTCTGC AACGAGCGCT TCACTCATTT GCCAGAGGCT 301GGGGGCCCGG AAGTCACGTA CGAGCCACCC CCGACAGCCC CCACCGGTGG 351TGGAACTCAC ACATGCCCAC CGTGCCCAGC ACCTGAACTC CTGGGGGGAC 401CGTCAGTCTT CCTCTTCCCC CCAAAACCCA AGGACACCCT CATGATCTCC 451CGGACCCCTG AGGTCACATG CGTGGTGGTG GACGTGAGCC ACGAAGACCC 501TGAGGTCAAG TTCAACTGGT ACGTGGACGG CGTGGAGGTG CATAATGCCA 551AGACAAAGCC GCGGGAGGAG CAGTACAACA GCACGTACCG TGTGGTCAGC 601GTCCTCACCG TCCTGCACCA GGACTGGCTG AATGGCAAGG AGTACAAGTG 651CAAGGTCTCC AACAAAGCCC TCCCAGCCCC CATCGAGAAA ACCATCTCCA 701AAGCCAAAGG GCAGCCCCGA GAACCACAGG TGTACACCCT GCCCCCATGC 751CGGGAGGAGA TGACCGAGAA CCAGGTCAGC CTGTGGTGCC TGGTCAAAGG 801CTTCTATCCC AGCGACATCG CCGTGGAGTG GGAGAGCAAT GGGCAGCCGG 851AGAACAACTA CAAGACCACG CCTCCCGTGC TGGACTCCGA CGGCTCCTTC 901TTCCTCTATA GCAAGCTCAC CGTGGACAAG AGCAGGTGGC AGCAGGGGAA 951CGTCTTCTCA TGCTCCGTGA TGCATGAGGC TCTGCACAAC CACTACACGC 1001AGGACAGCCT CTCCCTGTCT CCGGGT

The complementary form of ALK4-Fc fusion polypeptide (SEQ ID NO: 74) isas follows and may optionally be provided with lysine removed from theC-terminus.

(SEQ ID NO: 74) 1 MDAMKRGLCC VLLLCGAVFV SPGASGPRGV QALLCACTSC LQANYTCETD51 GACMVSIFNL DGMEHHVRTC IPKVELVPAG KPFYCLSSED LRNTHCCYTD 101YCNRIDLRVP SGHLKEPEHP SMWGPVETGG GTHTCPPCPA PELLGGPSVF 151LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP 201REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG 251QPREPQVCTL PPSREEMTKN QVSLSCAVKG FYPSDIAVEW ESRGQPENNY 301 KTTPPVLDSRGSFFLVSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL 351 SLSPGK

The leader sequence and the linker are underlined. To guide heterodimerformation with the ActRIIB-Fc fusion polypeptide of SEQ ID NOs: 70 and72 above, four amino acid substitutions (replacing a tyrosine with acysteine, a threonine with a serine, a leucine with an alanine, and atyrosine with a valine) can be introduced into the Fc domain of the ALK4fusion polypeptide as indicated by double underline above. To facilitatepurification of the ALK4-Fc:ActRIIB-Fc heterodimer, two amino acidsubstitutions (replacing an asparagine with an arginine and an aspartatewith an arginine) can also be introduced into the Fc domain of theALK4-Fc fusion polypeptide as indicated by double underline above. Theamino acid sequence of SEQ ID NO: 74 may optionally be provided withlysine removed from the C-terminus.

This ALK4-Fc fusion polypeptide is encoded by the following nucleic acid(SEQ ID NO: 75):

(SEQ ID NO: 75) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC51 AGTCTTCGTT TCGCCCGGCG CCTCCGGGCC CCGGGGGGTC CAGGCTCTGC 101TGTGTGCGTG CACCAGCTGC CTCCAGGCCA ACTACACGTG TGAGACAGAT 151GGGGCCTGCA TGGTTTCCAT TTTCAATCTG GATGGGATGG AGCACCATGT 201GCGCACCTGC ATCCCCAAAG TGGAGCTGGT CCCTGCCGGG AAGCCCTTCT 251ACTGCCTGAG CTCGGAGGAC CTGCGCAACA CCCACTGCTG CTACACTGAC 301TACTGCAACA GGATCGACTT GAGGGTGCCC AGTGGTCACC TCAAGGAGCC 351TGAGCACCCG TCCATGTGGG GCCCGGTGGA GACCGGTGGT GGAACTCACA 401CATGCCCACC GTGCCCAGCA CCTGAACTCC TGGGGGGACC GTCAGTCTTC 451CTCTTCCCCC CAAAACCCAA GGACACCCTC ATGATCTCCC GGACCCCTGA 501GGTCACATGC GTGGTGGTGG ACGTGAGCCA CGAAGACCCT GAGGTCAAGT 551TCAACTGGTA CGTGGACGGC GTGGAGGTGC ATAATGCCAA GACAAAGCCG 601CGGGAGGAGC AGTACAACAG CACGTACCGT GTGGTCAGCG TCCTCACCGT 651CCTGCACCAG GACTGGCTGA ATGGCAAGGA GTACAAGTGC AAGGTCTCCA 701ACAAAGCCCT CCCAGCCCCC ATCGAGAAAA CCATCTCCAA AGCCAAAGGG 751CAGCCCCGAG AACCACAGGT GTGCACCCTG CCCCCATCCC GGGAGGAGAT 801GACCAAGAAC CAGGTCAGCC TGTCCTGCGC CGTCAAAGGC TTCTATCCCA 851GCGACATCGC CGTGGAGTGG GAGAGCCGCG GGCAGCCGGA GAACAACTAC 901AAGACCACGC CTCCCGTGCT GGACTCCCGC GGCTCCTTCT TCCTCGTGAG 951CAAGCTCACC GTGGACAAGA GCAGGTGGCA GCAGGGGAAC GTCTTCTCAT 1001GCTCCGTGAT GCATGAGGCT CTGCACAACC ACTACACGCA GAAGAGCCTC 1051TCCCTGTCTC CGGGTAAA

The mature ALK4-Fc fusion polypeptide sequence is as follows (SEQ ID NO:76) and may optionally be provided with lysine removed from theC-terminus.

(SEQ ID NO: 76) 1 SGPRGVQALL CACTSCLQAN YTCETDGACM VSIFNLDGME HHVRTCIPKV51 ELVPAGKPFY CLSSEDLRNT HCCYTDYCNR IDLRVPSGHL KEPEHPSMWG 101PVETGGGTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD 151VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN 201GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVCTLPPSR EEMTKNQVSL 251SCAVKGFYPS DIAVEWESRG QPENNYKTTP PVLDSRGSFF LVSKLTVDKS 301RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK

This ALK4-Fc fusion polypeptide is encoded by the following nucleic acid(SEQ ID NO: 77):

(SEQ ID NO: 77) 1 TCCGGGCCCC GGGGGGTCCA GGCTCTGCTG TGTGCGTGCA CCAGCTGCCT51 CCAGGCCAAC TACACGTGTG AGACAGATGG GGCCTGCATG GTTTCCATTT 101TCAATCTGGA TGGGATGGAG CACCATGTGC GCACCTGCAT CCCCAAAGTG 151GAGCTGGTCC CTGCCGGGAA GCCCTTCTAC TGCCTGAGCT CGGAGGACCT 201GCGCAACACC CACTGCTGCT ACACTGACTA CTGCAACAGG ATCGACTTGA 251GGGTGCCCAG TGGTCACCTC AAGGAGCCTG AGCACCCGTC CATGTGGGGC 301CCGGTGGAGA CCGGTGGTGG AACTCACACA TGCCCACCGT GCCCAGCACC 351TGAACTCCTG GGGGGACCGT CAGTCTTCCT CTTCCCCCCA AAACCCAAGG 401ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC 451GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG TGGACGGCGT 501GGAGGTGCAT AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TACAACAGCA 551CGTACCGTGT GGTCAGCGTC CTCACCGTCC TGCACCAGGA CTGGCTGAAT 601GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT 651CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA CCACAGGTGT 701GCACCCTGCC CCCATCCCGG GAGGAGATGA CCAAGAACCA GGTCAGCCTG 751TCCTGCGCCG TCAAAGGCTT CTATCCCAGC GACATCGCCG TGGAGTGGGA 801GAGCCGCGGG CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGCTGG 851ACTCCCGCGG CTCCTTCTTC CTCGTGAGCA AGCTCACCGT GGACAAGAGC 901AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC ATGAGGCTCT 951GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG GGTAAA

ActRIIB-Fc and ALK4-Fc proteins of SEQ ID NO: 72 and SEQ ID NO: 76,respectively, may be co-expressed and purified from a CHO cell line, togive rise to a heteromeric complex comprising ALK4-Fc:ActRIIB-Fc.

Purification of various ALK4-Fc:ActRIIB-Fc complexes could be achievedby a series of column chromatography steps, including, for example,three or more of the following, in any order: protein A chromatography,Q sepharose chromatography, phenylsepharose chromatography, sizeexclusion chromatography, cation exchange chromatography, epitope-basedaffinity chromatography (e.g., with an antibody or functionallyequivalent ligand directed against an epitope on ALK4 or ActRIIB), andmultimodal chromatography (e.g., with resin containing bothelectrostatic and hydrophobic ligands). The purification could becompleted with viral filtration and buffer exchange.

In another approach to promote the formation of heteromultimer complexesusing asymmetric Fc fusion proteins, the Fc domains are altered tointroduce complementary hydrophobic interactions, an additionalintermolecular disulfide bond, and a histidine-to-arginine substitutionspecifically in the ActRIIB-Fc polypeptide chain for facilitatingpurification based on protein A affinity, as illustrated in theActRIIB-Fc polypeptide sequences of SEQ ID NOs: 78-81 and the ALK4-Fcpolypeptide sequences of SEQ ID NOs: 47, 48, 82, and 83. The ActRIIB-Fcfusion polypeptide and ALK4-Fc fusion polypeptide each employ the TPAleader: MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 38).

The ActRIIB-Fc polypeptide sequence (SEQ ID NO: 78) is shown below:

(SEQ ID NO: 78) 1 MDAMKRGLCC VLLLCGAVFV SPGASGRGEA ETRECIYYNA NWELERTNQS51 GLERCEGEQD KRLHCYASWR NSSGTIELVK KGCWLDDFNC YDRQECVATE 101ENPQVYFCCC EGNFCNERFT HLPEAGGPEV TYEPPPTAPT GGGTHTCPPC 151PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV 201DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP 251APIEKTISKA KGQPREPQVY TLPPCREEMT KNQVSLWCLV KGFYPSDIAV 301EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH 351EALHNRYTQK SLSLSPGK

The leader sequence and linker are underlined. To promote formation ofthe ALK4-Fc:ActRIIB-Fc heterodimer rather than either of the possiblehomodimeric complexes, two amino acid substitutions (replacing a serinewith a cysteine and a threonine with a trytophan) can be introduced intothe Fc domain of the ActRIIB-Fc fusion polypeptide as indicated bydouble underline above. Another amino acid substitution (replacinghistidine with arginine) can also be introduced into the Fc domain ofthe fusion protein as indicated by double underline above to facilitatepurification of the ALK4-Fc:ActRIIB-Fc heterodimer. The amino acidsequence of SEQ ID NO: 78 may optionally be provided with lysine removedfrom the C-terminus.

This ActRIIB-Fc fusion protein is encoded by the following nucleic acid(SEQ ID NO: 79):

(SEQ ID NO: 79) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC51 AGTCTTCGTT TCGCCCGGCG CCTCTGGGCG TGGGGAGGCT GAGACACGGG 101AGTGCATCTA CTACAACGCC AACTGGGAGC TGGAGCGCAC CAACCAGAGC 151GGCCTGGAGC GCTGCGAAGG CGAGCAGGAC AAGCGGCTGC ACTGCTACGC 201CTCCTGGCGC AACAGCTCTG GCACCATCGA GCTCGTGAAG AAGGGCTGCT 251GGCTAGATGA CTTCAACTGC TACGATAGGC AGGAGTGTGT GGCCACTGAG 301GAGAACCCCC AGGTGTACTT CTGCTGCTGT GAAGGCAACT TCTGCAACGA 351GCGCTTCACT CATTTGCCAG AGGCTGGGGG CCCGGAAGTC ACGTACGAGC 401CACCCCCGAC AGCCCCCACC GGTGGTGGAA CTCACACATG CCCACCGTGC 451CCAGCACCTG AACTCCTGGG GGGACCGTCA GTCTTCCTCT TCCCCCCAAA 501ACCCAAGGAC ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG 551TGGTGGACGT GAGCCACGAA GACCCTGAGG TCAAGTTCAA CTGGTACGTG 601GACGGCGTGG AGGTGCATAA TGCCAAGACA AAGCCGCGGG AGGAGCAGTA 651CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTG CACCAGGACT 701GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCA 751GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC 801ACAGGTGTAC ACCCTGCCCC CATGCCGGGA GGAGATGACC AAGAACCAGG 851TCAGCCTGTG GTGCCTGGTC AAAGGCTTCT ATCCCAGCGA CATCGCCGTG 901GAGTGGGAGA GCAATGGGCA GCCGGAGAAC AACTACAAGA CCACGCCTCC 951CGTGCTGGAC TCCGACGGCT CCTTCTTCCT CTATAGCAAG CTCACCGTGG 1001ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCAT 1051GAGGCTCTGC ACAACCGCTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG 1101 TAAA

The mature ActRIIB-Fc fusion polypeptide is as follows (SEQ ID NO: 80)and may optionally be provided with lysine removed from the C-terminus.

(SEQ ID NO: 80) 1XGRGEAETREC IYYNANWELE RTNQSGLERC EGEQDKRLHC YASWRNSSGT 51IELVKKGCWL DDFNCYDRQE CVATEENPQV YFCCCEGNFC NERFTHLPEA 101GGPEVTYEPP PTAPTGGGTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS 151RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS 201VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPC 251REEMTKNQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF 301FLYSKLTVDK SRWQQGNVFS CSVMHEALHN RYTQKSLSLS PGK

This ActRIIB-Fc fusion polypeptide is encoded by the following nucleicacid (SEQ ID NO: 81):

(SEQ ID NO: 81) 1 GGGCGTGGGG AGGCTGAGAC ACGGGAGTGC ATCTACTACA ACGCCAACTG51 GGAGCTGGAG CGCACCAACC AGAGCGGCCT GGAGCGCTGC GAAGGCGAGC 101AGGACAAGCG GCTGCACTGC TACGCCTCCT GGCGCAACAG CTCTGGCACC 151ATCGAGCTCG TGAAGAAGGG CTGCTGGCTA GATGACTTCA ACTGCTACGA 201TAGGCAGGAG TGTGTGGCCA CTGAGGAGAA CCCCCAGGTG TACTTCTGCT 251GCTGTGAAGG CAACTTCTGC AACGAGCGCT TCACTCATTT GCCAGAGGCT 301GGGGGCCCGG AAGTCACGTA CGAGCCACCC CCGACAGCCC CCACCGGTGG 351TGGAACTCAC ACATGCCCAC CGTGCCCAGC ACCTGAACTC CTGGGGGGAC 401CGTCAGTCTT CCTCTTCCCC CCAAAACCCA AGGACACCCT CATGATCTCC 451CGGACCCCTG AGGTCACATG CGTGGTGGTG GACGTGAGCC ACGAAGACCC 501TGAGGTCAAG TTCAACTGGT ACGTGGACGG CGTGGAGGTG CATAATGCCA 551AGACAAAGCC GCGGGAGGAG CAGTACAACA GCACGTACCG TGTGGTCAGC 601GTCCTCACCG TCCTGCACCA GGACTGGCTG AATGGCAAGG AGTACAAGTG 651CAAGGTCTCC AACAAAGCCC TCCCAGCCCC CATCGAGAAA ACCATCTCCA 701AAGCCAAAGG GCAGCCCCGA GAACCACAGG TGTACACCCT GCCCCCATGC 751CGGGAGGAGA TGACCAAGAA CCAGGTCAGC CTGTGGTGCC TGGTCAAAGG 801CTTCTATCCC AGCGACATCG CCGTGGAGTG GGAGAGCAAT GGGCAGCCGG 851AGAACAACTA CAAGACCACG CCTCCCGTGC TGGACTCCGA CGGCTCCTTC 901TTCCTCTATA GCAAGCTCAC CGTGGACAAG AGCAGGTGGC AGCAGGGGAA 951CGTCTTCTCA TGCTCCGTGA TGCATGAGGC TCTGCACAAC CGCTACACGC 1001AGAAGAGCCT CTCCCTGTCT CCGGGTAAA

The complementary form of ALK4-Fc fusion polypeptide is SEQ ID NO: 47(shown above), which contains four amino acid substitutions to guideheterodimer formation with the ActRIIB-Fc fusion polypeptide of SEQ IDNOs: 78 and 80 and may optionally be provided with lysine removed fromthe C-terminus.

This ALK4-Fc fusion polypeptide is encoded by the following nucleic acid(SEQ ID NO: 82):

(SEQ ID NO: 82) 1 ATGGATGCAA TGAAGAGAGG GCTCTGCTGT GTGCTGCTGC TGTGTGGAGC51 AGTCTTCGTT TCGCCCGGCG CCTCCGGGCC CCGGGGGGTC CAGGCTCTGC 101TGTGTGCGTG CACCAGCTGC CTCCAGGCCA ACTACACGTG TGAGACAGAT 151GGGGCCTGCA TGGTTTCCAT TTTCAATCTG GATGGGATGG AGCACCATGT 201GCGCACCTGC ATCCCCAAAG TGGAGCTGGT CCCTGCCGGG AAGCCCTTCT 251ACTGCCTGAG CTCGGAGGAC CTGCGCAACA CCCACTGCTG CTACACTGAC 301TACTGCAACA GGATCGACTT GAGGGTGCCC AGTGGTCACC TCAAGGAGCC 351TGAGCACCCG TCCATGTGGG GCCCGGTGGA GACCGGTGGT GGAACTCACA 401CATGCCCACC GTGCCCAGCA CCTGAACTCC TGGGGGGACC GTCAGTCTTC 451CTCTTCCCCC CAAAACCCAA GGACACCCTC ATGATCTCCC GGACCCCTGA 501GGTCACATGC GTGGTGGTGG ACGTGAGCCA CGAAGACCCT GAGGTCAAGT 551TCAACTGGTA CGTGGACGGC GTGGAGGTGC ATAATGCCAA GACAAAGCCG 601CGGGAGGAGC AGTACAACAG CACGTACCGT GTGGTCAGCG TCCTCACCGT 651CCTGCACCAG GACTGGCTGA ATGGCAAGGA GTACAAGTGC AAGGTCTCCA 701ACAAAGCCCT CCCAGCCCCC ATCGAGAAAA CCATCTCCAA AGCCAAAGGG 751CAGCCCCGAG AACCACAGGT GTGCACCCTG CCCCCATCCC GGGAGGAGAT 801GACCAAGAAC CAGGTCAGCC TGTCCTGCGC CGTCAAAGGC TTCTATCCCA 851GCGACATCGC CGTGGAGTGG GAGAGCAATG GGCAGCCGGA GAACAACTAC 901AAGACCACGC CTCCCGTGCT GGACTCCGAC GGCTCCTTCT TCCTCGTGAG 951CAAGCTCACC GTGGACAAGA GCAGGTGGCA GCAGGGGAAC GTCTTCTCAT 1001GCTCCGTGAT GCATGAGGCT CTGCACAACC ACTACACGCA GAAGAGCCTC 1051TCCCTGTCTC CGGGTAAA

The mature ALK4-Fc fusion polypeptide sequence is SEQ ID NO: 48 (shownabove) and may optionally be provided with lysine removed from theC-terminus.

This ALK4-Fc fusion polypeptide is encoded by the following nucleic acid(SEQ ID NO: 83):

(SEQ ID NO: 83) 1 TCCGGGCCCC GGGGGGTCCA GGCTCTGCTG TGTGCGTGCA CCAGCTGCCT51 CCAGGCCAAC TACACGTGTG AGACAGATGG GGCCTGCATG GTTTCCATTT 101TCAATCTGGA TGGGATGGAG CACCATGTGC GCACCTGCAT CCCCAAAGTG 151GAGCTGGTCC CTGCCGGGAA GCCCTTCTAC TGCCTGAGCT CGGAGGACCT 201GCGCAACACC CACTGCTGCT ACACTGACTA CTGCAACAGG ATCGACTTGA 251GGGTGCCCAG TGGTCACCTC AAGGAGCCTG AGCACCCGTC CATGTGGGGC 301CCGGTGGAGA CCGGTGGTGG AACTCACACA TGCCCACCGT GCCCAGCACC 351TGAACTCCTG GGGGGACCGT CAGTCTTCCT CTTCCCCCCA AAACCCAAGG 401ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC 451GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG TGGACGGCGT 501GGAGGTGCAT AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TACAACAGCA 551CGTACCGTGT GGTCAGCGTC CTCACCGTCC TGCACCAGGA CTGGCTGAAT 601GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC AAAGCCCTCC CAGCCCCCAT 651CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA CCACAGGTGT 701GCACCCTGCC CCCATCCCGG GAGGAGATGA CCAAGAACCA GGTCAGCCTG 751TCCTGCGCCG TCAAAGGCTT CTATCCCAGC GACATCGCCG TGGAGTGGGA 801GAGCAATGGG CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGCTGG 851ACTCCGACGG CTCCTTCTTC CTCGTGAGCA AGCTCACCGT GGACAAGAGC 901AGGTGGCAGC AGGGGAACGT CTTCTCATGC TCCGTGATGC ATGAGGCTCT 951GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG GGTAAA

ActRIIB-Fc and ALK4-Fc proteins of SEQ ID NO: 80 and SEQ ID NO: 48,respectively, may be co-expressed and purified from a CHO cell line, togive rise to a heteromeric complex comprising ALK4-Fc:ActRIIB-Fc.

Purification of various ALK4-Fc:ActRIIB-Fc complexes could be achievedby a series of column chromatography steps, including, for example,three or more of the following, in any order: protein A chromatography,Q sepharose chromatography, phenylsepharose chromatography, sizeexclusion chromatography and epitope-based affinity chromatography(e.g., with an antibody or functionally equivalent ligand directedagainst an epitope on ALK4 or ActRIIB), and multimodal chromatography(e.g., with resin containing both electrostatic and hydrophobicligands). The purification could be completed with viral filtration andbuffer exchange.

Example 2. Ligand Binding Profile of ALK4-Fc:ActRIIB-Fc HeterodimerCompared to ActRIIB-Fc Homodimer and ALK4-Fc Homodimer

A Biacore™-based binding assay was used to compare ligand bindingselectivity of the ALK4-Fc:ActRIIB-Fc heterodimeric complex describedabove with that of ActRIIB-Fc and ALK4-Fc homodimer complexes. TheALK4-Fc:ActRIIB-Fc heterodimer, ActRIIB-Fc homodimer, and ALK4-Fchomodimer were independently captured onto the system using an anti-Fcantibody. Ligands were injected and allowed to flow over the capturedreceptor protein. Results are summarized in the table below, in whichligand off-rates (k_(d)) most indicative of effective ligand traps aredenoted by bold text.

Ligand binding profile of ALK4-Fc:ActRIIB-Fc heterodimer compared toActRIIB-Fc homodimer and ALK4-Fc homodimer ActRIIB-Fc ALK4-FcALK4-Fc:ActRIIB-Fc homodimer homodimer heterodimer k_(a) k_(d) K_(D)k_(a) ka K_(D) k_(a) k_(d) K_(D) Ligand (1/Ms) (1/s) (pM) (1/Ms) (1/s)(pM) (1/Ms) (1/s) (pM) Activin A 1.2 × 10⁷ 2.3 × 10⁻⁴ 19 5.8 × 10⁵ 1.2 ×10⁻² 20000  1.3 × 10⁷ 1.5 × 10⁻⁴ 12 Activin B 5.1 × 10⁶ 1.0 × 10⁻⁴ 20 Nobinding 7.1 × 10⁶ 4.0 × 10⁻⁵ 6 BMP6 3.2 × 10⁷ 6.8 × 10⁻³ 190 — 2.0 × 10⁶5.5 × 10⁻³ 2700 BMP9 1.4 × 10⁷ 1.1 × 10⁻³ 77 — Transient* 3400 BMP10 2.3× 10⁷ 2.6 × 10⁻⁴ 11 — 5.6 × 10⁷ 4.1 × 10⁻³ 74 GDF3 1.4 × 10⁶ 2.2 × 10⁻³1500 — 3.4 × 10⁶ 1.7 × 10⁻² 4900 GDF8 8.3 × 10⁵ 2.3 × 10⁻⁴ 280 1.3 × 10⁵1.9 × 10⁻³ 15000† 3.9 × 10⁵ 2.1 × 10⁻⁴ 550 GDF11 5.0 × 10⁷ 1.1 × 10⁻⁴ 25.0 × 10⁶ 4.8 × 10⁻³  270† 3.8 × 10⁷ 1.1 × 10⁻⁴ 3 *Indeterminate due totransient nature of interaction †Very low signal — Not tested

These comparative binding data demonstrate that ALK4-Fc:ActRIIB-Fcheterodimer has an altered binding profile/selectivity relative toeither ActRIIB-Fc or ALK4-Fc homodimers. ALK4-Fc:ActRIIB-Fc heterodimerdisplays enhanced binding to activin B compared with either homodimer,retains strong binding to activin A, GDF8, and GDF11 as observed withActRIIB-Fc homodimer, and exhibits substantially reduced binding toBMP9, BMP10, and GDF3. In particular, BMP9 displays low or no observableaffinity for ALK4-Fc:ActRIIB-Fc heterodimer, whereas this ligand bindsstrongly to ALK4-Fc:ActRIIB-Fc heterodimer. Like the ActRIIB-Fchomodimer, the heterodimer retains intermediate-level binding to BMP6.See FIG. 4.

In addition, an A-204 Reporter Gene Assay was used to evaluate theeffects of ALK4-Fc:ActRIIB-Fc heterodimer and ActRIIB-Fc:ActRIIB-Fchomodimer on signaling by activin A, activin B, GDF11, GDF8, BMP10, andBMP9. Cell line: Human Rhabdomyosarcoma (derived from muscle). Reportervector: pGL3(CAGA)12 (as described in Dennler et al, 1998, EMBO 17:3091-3100). The CAGA12 motif is present in TGF-beta responsive genes(PAI-1 gene), so this vector is of general use for factors signalingthrough Smad2 and 3. An exemplary A-204 Reporter Gene Assay is outlinedbelow.

-   -   Day 1: Split A-204 cells into 48-well plate.    -   Day 2: A-204 cells transfected with 10 ug pGL3(CAGA)12 or        pGL3(CAGA)12(10 ug)+pRLCMV (1 ug) and Fugene.    -   Day 3: Add factors (diluted into medium+0.1% BSA). Inhibitors        need to be pre-incubated with Factors for about one hr before        adding to cells. About six hrs later, cells are rinsed with PBS        and then lysed.

Following the above steps, applicant performed a Luciferase assay.

Both the ALK4-Fc:ActRIIB-Fc heterodimer and ActRIIB-Fc:ActRIIB-Fchomodimer were determined to be potent inhibitors of activin A, activinB, GDF11, and GDF8 in this assay. In particular, as can be seen in thecomparative homodimer/heterodimer IC₅₀ data illustrated in FIG. 7,ALK4-Fc:ActRIIB-Fc heterodimer inhibits activin A, activin B, GDF8, andGDF11 signaling pathways similarly to the ActRIIB-Fc:ActRIIB-Fchomodimer. However, ALK4-Fc:ActRIIB-Fc heterodimer inhibition of BMP9and BMP10 signaling pathways is significantly reduced compared to theActRIIB-Fc:ActRIIB-Fc homodimer. This data is consistent with theabove-discussed binding data in which it was observed that both theALK4-Fc:ActRIIB-Fc heterodimer and ActRIIB-Fc:ActRIIB-Fc homodimerdisplay strong binding to activin A, activin B, GDF8, and GDF11, butBMP10 and BMP9 have significantly reduced affinity for theALK4-Fc:ActRIIB-Fc heterodimer compared to the ActRIIB-Fc:ActRIIB-Fchomodimer.

Together, these data therefore demonstrate that ALK4-Fc:ActRIIB-Fcheterodimer is a more selective antagonist of activin B, activin A,GDF8, and GDF11 compared to ActRIIB-Fc homodimer. Accordingly, anALK4-Fc:ActRIIB-Fc heterodimer will be more useful than an ActRIIB-Fchomodimer in certain applications where such selective antagonism isadvantageous. Examples include therapeutic applications where it isdesirable to retain antagonism of one or more of activin A, activin B,activin AB, GDF8, and GDF11 but minimize antagonism of one or more ofBMP9, BMP10, GDF3, and BMP6.

Example 3. Activity Profile of ALK4-Fc:ActRIIB-Fc Heterodimer in MiceCompared to ActRIIB-Fc Homodimer

Homodimeric and heterodimeric complexes were tested in mice toinvestigate differences in their activity profiles in vivo. Wild-typeC57BL/6 mice were dosed subcutaneously with ActRIIB-Fc homodimer (10mg/kg), ALK4-Fc:ActRIIB-Fc heterodimer (3 or 10 mg/kg), or vehicle(phosphate-buffered saline, PBS) twice per week for 4 weeks beginning atapproximately 10 weeks of age (n=9 mice per group). ALK4-Fc homodimerwas not tested in vivo due to its inability to bind ligands with highaffinity under cell-free conditions as determined by surface plasmonresonance. Study endpoints included: body weight; total lean mass andtotal adipose mass as determined by nuclear magnetic resonance (NMR) atbaseline and study completion (4 weeks); total bone mineral density asdetermined by dual energy x-ray absorptiometry (DEXA) at baseline and 4weeks; and weights of the gastrocnemius, rectus femoris, and pectoralismuscles determined at 4 weeks.

Activity of ActRIIB-Fc and ALK4-Fc Complexes in Wild-Type MiceActRIIB-Fc ALK4-Fc:ActRIIB-Fc Endpoint homodimer heterodimer (4 wk)Vehicle 10 mg/kg 10 mg/kg 3 mg/kg Change in body weight ↑15%  ↑38%**↑41%** ↑33%** from baseline Change in total lean mass ↓1%  ↑5%**  ↑5%** ↑5%** from baseline Change in total adipose ↑5% ↓3.6%**  ↓3.5%** ↓3.5%**  mass from baseline Change in total bone ↑8% ↑14%*  ↑12%*  ↑11% mineral density from baseline Gastrocnemius weight† 23 36** 35** 30**Femoris weight† 11.5 17** 16** 14** Pectoralis weight† 15 23** 28** 23***P < 0.05 vs. vehicle **P < 0.01 vs. vehicle †Combined left and rightmuscle weights normalized to femur length (mg/mm) to control for bodysize

Study results are summarized in the table above. As expected, ActRIIB-Fchomodimer caused marked changes in body composition, many consistentwith known effects of GDF8 and activin inhibition. Treatment ofwild-type mice with ActRIIB-Fc homodimer more than doubled body weightgain over the course of the study compared to vehicle-treated controls.Accompanying this net weight gain were significant increases in totallean mass and total bone mineral density, as well as a significantreduction in total adipose mass, compared to vehicle. It should berecognized that normalized (percentage-based) changes in lean andadipose tissues differ in their correspondence to absolute changesbecause lean mass (typically about 70% of body weight in a mouse) ismuch larger than adipose mass (typically about 10% of body weight).Individual skeletal muscles examined, including the gastrocnemius,femoris, and pectoralis all increased significantly in weight comparedto vehicle controls over the course of treatment with ActRIIB-Fchomodimer.

The ALK4-Fc:ActRIIB-Fc heterodimer produced certain effects strikinglysimilar to those of the ActRIIB-Fc homodimer despite differential ligandselectivity of the two complexes. As shown in the table above, treatmentof mice with the ALK4-Fc:ActRIIB-Fc heterodimer at a dose level of 10mg/kg matched, nearly matched, or exceeded the effects of ActRIIB-Fchomodimer at the same dose level for all endpoints listed. Effects ofthe ALK4-Fc:ActRIIB-Fc heterodimer at 3 mg/kg were mildly attenuated forseveral endpoints compared to 10 mg/kg, thus providing evidence for adose-effect relationship.

Thus, an ALK4-Fc:ActRIIB-Fc heterodimer exerts beneficial anaboliceffects on skeletal muscle and bone, and catabolic effects on adiposetissue, very similar to those of ActRIIB-Fc homodimer. However, unlikeActRIIB homodimer, ALK4-Fc:ActRIIB-Fc heterodimer exhibits onlylow-affinity or transient binding to BMP9 and BMP10 and so should havelittle to no concurrent inhibition on processes mediated by thoseligands, such as angiogenesis. This novel selectivity will be useful,for example, in treating patients in need of stimulatory effects onmuscle and bone, and inhibitory effects on fat, but not in need ofaltered angiogenesis.

Example 4. ALK4:ActRIIB Heteromultimer Treatment Suppresses KidneyFibrosis and Inflammation and Reduces Kidney Injury

The effects of the ALK4-Fc:ActRIIB-Fc heterodimer described in Example 2on kidney disease was assessed in a mouse unilateral ureteralobstruction model. See, e.g., Klahr and Morrissey (2002) Am J PhysiolRenal Physiol 283: F861-F875.

Twenty-four C57BL/6 male mice 12 weeks of age underwent left unilateralureteral ligation twice at the level of the lower pole of kidney. After3 days, eight mice were euthanized and kidneys from individual animalswere harvested to assess kidney injury. The remaining mice wererandomized into two groups: i) eight mice were injected subcutaneouslywith the ALK4-Fc:ActRIIB-Fc heterodimer at a dose of 10 mg/kg at day 3,day 7, day 10, and day 14 after surgery and a ii) eight mice wereinjected subcutaneously with vehicle control, phosphate buffered saline(PBS), at day 3, day 7, day 10, and day 14 after surgery. Both groupswere sacrificed at day 17 in accordance with the relevant Animal CareGuidelines. Half kidneys from individual animals were collected forhistology analysis (H&E, and Masson's Trichrome stain), from both theUUO kidney and contralateral kidney, and ¼ kidneys were used for RNAextraction (RNeasy Midi Kit, Qiagen, IL).

Gene expression analysis on UUO kidney samples was performed to assesslevels of various genes. QRT-PCR was performed on a CFX Connect™Real-time PCR detection system (Bio-Rad, CA) to evaluate the expressionof various fibrotic genes (Col1a1, Fibronectin, PAI-1, CTGF, and a-SMA),inflammatory genes (TNFa, and MCP1), cytokines (TGFβ1, TGFβ2, TGFβ3, andactivin A), and kidney injury genes (NGAL. See FIG. 8. Treatment of micewith ALK4-Fc:ActRIIB-Fc heterodimer significantly suppressed theexpression of fibrotic and inflammatory genes, inhibited theupregulation of TGFβ 1/2/3 and reduced kidney injury. Histology dataconfirmed that ALK4-Fc:ActRIIB-Fc heterodimer treatment significantlyinhibited kidney fibrosis and reduced kidney injury in the UUO model.

Together, these data demonstrate that ALK4:ActRIIB heteromultimertreatment suppresses kidney fibrosis and inflammation and reduces kidneyinjury. Moreover, these data indicate that other ALK4:ActRIIBantagonists may be useful in the treatment or preventing of kidneydisease including, for example, antagonists of ALK4 and/orActRIIB-binding ligands, antagonists of ALK4 and/or ActRIIB receptors,antagonists of ALK4 and/or ActRIIB downstream signaling mediators (e.g.,Smads), and antagonists of TGFβ superfamily co-receptors associated withALK4 and/or ActRIIB.

Example 5. ALK4:ActRIIB Heteromultimer Treatment Suppresses KidneyFibrosis and Reduces Proteinuria in Alport Mouse Models

The effects of the ALK4-Fc:ActRIIB-Fc heterodimer described in Example 2on kidney disease was assessed in Col4a3−/− and Col4a5 Alport mousemodels. See, e.g., Cosgrove D et al, (1996) Genes Dev. 10(23): 2981-92;Rheault M N et al, (2004) J Am Soc Nephrol. 15(6): 1466-74.

Sixteen Col4a3−/− male mice 2 weeks of age were randomized into twogroups: i) eight mice were biweekly injected subcutaneously with theALK4-Fc:ActRIIB-Fc heterodimer at a dose of 10 mg/kg for 5 weeks ii)eight mice were injected subcutaneously with vehicle control, phosphatebuffered saline (PBS) for 5 weeks. Urine samples from individual animalswere collected by metabolic cages at 4 weeks, 5 weeks, 6 weeks, and 7weeks of age respectively. Both groups were sacrificed at 7 weeks of ageand kidneys from individual animals were harvested to assess kidneyinjury. Half kidneys from individual animals were collected forhistology analysis (H&E, and Masson's Trichrome stain), and urinesamples from individual animals were used to evaluate albuminuria, ahallmark of glomerular damage in Alport syndrome (Mouse Albumin AntigenAssay, Molecular Innovation, MI; QuantiChrom Creatinine Assay Kit,BioAssay System, CA).

Albuminuria analysis was performed to assess levels of albumin in urineafter normalized to urine creatinine (ACR: albumin-to-creatinine ratio).Treatment of mice with ALK4-Fc:ActRIIB-Fc heterodimer significantlyreduced albuminuria (FIG. 9A) and suppressed extracellular matrixdeposition measured by immunofluorescent staining of Collagen-I (FIG.10A). In line with the albuminuria result, histology data confirmed thatALK4-Fc:ActRIIB-Fc heterodimer treatment significantly inhibited kidneyfibrosis (FIG. 10B) and reduced sclerotic glomeruli (FIG. 10C) inCol4a3−/− mice.

To confirm the suppression of albuminuria in Alport mouse model,ALK4-Fc:ActRIIB-Fc heterodimer was evaluated in Col4a5 X-linked Alportmouse model. Thirty Col4a5 hemizygous male mice 17 weeks of age wererandomized into two groups: i) fifteen mice were biweekly injectedsubcutaneously with the ALK4-Fc:ActRIIB-Fc heterodimer at a dose of 10mg/kg for 12 weeks ii) fifteen mice were injected subcutaneously withvehicle control, phosphate buffered saline (PBS) for 12 weeks. Urinesamples from individual animals were collected by metabolic cages at 17weeks, 20 weeks, 23 weeks, 26 weeks, and 29 weeks of age respectivelyfor albuminuria analysis. Consistent with the result in Col4a3−/− mice,treatment of Col4a5 hemizygous mice with ALK4-Fc:ActRIIB-Fc heterodimersignificantly reduced albuminuria (FIG. 9B).

Together, these data demonstrate that ALK4:ActRIIB heterodimer treatmentsuppresses kidney fibrosis and reduces sclerotic glomeruli, associatedwith the improvement of albuminuria in Alport mouse models. Moreover,these data indicate that other ALK4:ActRIIB antagonists may be useful inthe treatment or preventing of kidney dysfunction in Alport syndrome.

Example 6. ALK4:ActRIIB Heteromultimer Treatment Improves Muscle Massand Strength in a Mouse Model of Amyotrophic Lateral Sclerosis (ALS)

The effects of the ALK4-Fc:ActRIIB-Fc heterodimer described in Example 2on muscle mass and strength in a disease condition was assessed in theSOD1 mouse model of amyotrophic lateral sclerosis (ALS). See, e.g.,Gurney et al. (1994) Science 264(5166): 1772-1775. In the SOD1 model,mice develop mild disease symptoms, muscle weakness and/or stiffness,around 12 weeks of age, and they develop more severe symptoms, muscleparalysis and/or respiratory failure, around 16 weeks of age. In thisstudy, SOD1 mice were examined at 8 weeks (“pre-symptomatic” stage), 12weeks (“disease-onset” stage), and 16 weeks (“disease-progression”stage) of age.

SOD1 [B6SJL-Tg(SOD1*G93A)1Gur/J] mice at 5 weeks of age were separatedinto separate groups: i) mice subcutaneously injected with vehiclecontrol, phosphate buffered saline, twice weekly; and ii) micesubcutaneously injected with the ALK4-Fc:ActRIIB-Fc heterodimer at adose of 10 mg/kg twice weekly. These two treatment groups were alsocompared to wild-type mice at 5 weeks of age, receiving subcutaneousinjects of vehicle control, phosphate buffered saline, twice weekly.Mice were observed for changes in muscle mass, muscle fiber area, andchanges in muscle tetanic force over the course of 11 weeks. Compared towild-type mice, SOD1 mice receiving vehicle displayed significantlydecreased tibialis anterior (TA) muscle mass at 12 weeks (approximately55 mg vs. 40 mg muscle mass) and 16 weeks approximately 60 mg vs. 35 mgmuscle mass) of age. In contrast, SOD1 mice receiving ALK4-Fc:ActRIIB-Fcsurprisingly displayed significantly TA more muscle mass than wild-typemice at 8 weeks (approximately 55 mg vs. 80 mg muscle mass) and 12 weeks(approximately 60 mg vs. 85 mg muscle mass) of age. Indisease-progression stage mice, SOD1 mice receiving ALK4-Fc:ActRIIB-Fcdisplayed slightly reduced TA muscle mass compared to wild-type animals,but ALK4-Fc:ActRIIB-Fc treated animals had significantly more (>80%) TAmuscle mass compared to SOD1 mice receiving vehicle. Similar trends wereobserved upon examining TA muscle fiber area (μm²). In particular,compared to wild-type mice, SOD1 mice receiving vehicle displayed slightreductions in TA muscle fiber at 12 weeks of age (approximately 1900 μm²vs. 1700 μm² muscle fiber), and dramatic reductions in TA muscle fiberwere observed in disease-progression stage mice (approximately 2200 μm²vs. 1450 μm² muscle fiber). In contrast, SOD1 mice receivingALK4-Fc:ActRIIB-Fc displayed significantly more TA muscle mass thanwild-type mice 12 weeks of age (approximately 2300 μm² vs. 1900 μm²muscle fiber). In disease-progression stage mice, SOD1 mice receivingALK4-Fc:ActRIIB-Fc displayed slightly reduced TA muscle fiberconcentration compared to wild-type animals, but ALK4-Fc:ActRIIB-Fctreated animals had significantly (>29%) more TA muscle fiberconcentration compared to SOD1 mice receiving vehicle. The increase inTA muscle mass and fiber content correlated with an increase instrength. For example, compared to wild-type mice, SOD1 mice receivingvehicle displayed moderately decreased TA muscle strength, measured bypeak tetanic force (mN) of the muscle, at 12 weeks of age (approximately1300 mN vs. 900 mN peak tetanic force), and dramatic reductions in TAmuscle strength were observed in disease-progression stage mice(approximately 1250 mN vs. 600 mN peak tetanic force). SOD1 micereceiving ALK4-Fc:ActRIIB-Fc displayed significantly increased TA musclestrength than SOD1 mice receiving vehicle at 12 weeks of age (>25%increased muscle strength) and 16 weeks of age (>43% increased musclestrength).

Taken together, the data demonstrate that ALK4-mFc:ActRIIB-mFc therapyis able to increase muscle mass and strength in a mouse model of ALS.Therefore, the data indicate that ALK4-mFc:ActRIIB-mFc, and potentiallyother ALK4:ActRIIB antagonists, may be used to treat other muscledisorders, particularly motor neuron and neuromuscular diseases.

Example 7. ALK4:ActRIIB Heteromultimer Treatment Improves DiaphragmStrength in a Mouse Model of Muscular Dystrophy

The effects of the ALK4-Fc:ActRIIB-Fc heterodimer described in Example 2on muscle mass and strength in a disease condition was further assessedin the mdx mouse model of muscular dystropy. In this mdx model, thephenotype of dystrophin deficiency can be observed at any early age(i.e., around 7 weeks of age).

Mdx (D2.B10-Dmd^(mdx)/J) mice at 5 weeks of age were separated intoseparate groups: i) mice subcutaneously injected with vehicle control,phosphate buffered saline, twice weekly; and ii) mice subcutaneouslyinjected with the ALK4-Fc:ActRIIB-Fc heterodimer at a dose of 10 mg/kgtwice weekly. These two treatment groups were also compared to wild-typemice at 5 weeks of age, receiving subcutaneous injects of vehiclecontrol, phosphate buffered saline, twice weekly. Mice were observed forchanges in muscle strength after 8 weeks of treatment. Compared towild-type mice, mdx mice receiving vehicle displayed a significantdecreased in diaphragm muscle strength, measured by specific force (kPa)of the muscle, after 8 weeks [i.e., approximately 89 kPa (wild-type) vs.32 kPa (mdx)]. In contrast, ALK4-Fc:ActRIIB-Fc treatment was observed tosignificantly increase diaphragm strength (>63%) compared to mdx micereceiving vehicle control.

The data demonstrate that ALK4-mFc:ActRIIB-mFc therapy is able toincrease strength in a mouse model of mdx. Therefore, the data indicatethat ALK4-mFc:ActRIIB-mFc, and potentially other ALK4:ActRIIBantagonists, may be used to treat other muscle disorders, particularlymuscular dystrophies including, for example, DMD and BMD.

We claim:
 1. A recombinant ALK4:ActRIIB heteromultimer comprising atleast one ALK4-Fc fusion protein and at least one ActRIIB-Fc fusionprotein, wherein the ALK4-Fc fusion protein comprises the amino acidsequence of SEQ ID NO: 76, and wherein the ActRIIB-Fc fusion proteincomprises the amino acid sequence of SEQ ID NO: 72.